Non-random distribution of instability-associated chromosomal rearrangement breakpoints in human lymphoblastoid cells

Genomic instability is observed in tumors and in a large fraction of the progeny surviving irradiation. One of the best-characterized phenotypic manifestations of genomic instability is delayed chromosome aberrations. Our working hypothesis for the current study was that if genomic instability is in part attributable to cis mechanisms, we should observe a non-random distribution of chromosomes or sites involved in instability-associated rearrangements, regardless of radiation quality, dose, or trans factor expression. We report here the karyotypic examination of 296 instability-associated chromosomal rearrangement breaksites (IACRB) from 118 unstable TK6 human B lymphoblast, and isogenic derivative, clones. When we tested whether IACRB were distributed across the chromosomes based on target size, a significant non-random distribution was evident (p < 0.00001), and three IACRB hotspots (chromosomes 11, 12, and 22) and one IACRB coldspot (chromosome 2) were identified. Statistical analysis at the chromosomal band-level identified four IACRB hotspots accounting for 20% of all instability-associated breaks, two of which account for over 14% of all IACRB. Further, analysis of independent clones provided evidence within 14 individual clones of IACRB clustering at the chromosomal band level, suggesting a predisposition for further breaks after an initial break at some chromosomal bands. All of these events, independently, or when taken together, were highly unlikely to have occurred by chance (p < 0.000001). These IACRB band-level cluster hotspots were observed independent of radiation quality, dose, or cellular p53 status. The non-random distribution of instability-associated chromosomal rearrangements described here significantly differs from the distribution that was observed in a first-division post-irradiation metaphase analysis (p = 0.0004). Taken together, these results suggest that genomic instability may be in part driven by chromosomal cis mechanisms.     

Influence of PAHs in ambient air on chromosomal aberrations in exposed subjects: international study - EXPAH

The aim of this study was to determine the influence of carcinogenic polycyclic aromatic hydrocarbons (c–PAHs) in complex mixtures in ambient air on DNA damage (chromosomal aberrations) in occupationally exposed subjects measured as percent of aberrant cells (% AB.C.).

There were in total 203 exposed subjects and 150 respective controls in the whole project, allocated in three different European cities – Kosice (Slovakia), Prague (Czech Republic) and Sofia (Bulgaria). The studied population from Kosice (Slovakia) consisted of 106 subjects. From these 51 were exposed policemen and 55 were controls. The Czech population comprised 52 exposed policemen and 50 controls. In Bulgaria, there were two equally numerous exposed groups: 50 policemen and 50 professional bus drivers together with 45 controls. According to personal monitoring, policemen and bus drivers in the Bulgarian capital Sofia were exposed to the highest levels of c-PAHs amongst the exposed subject groups in the cities (45.3 ± 25.9 ng/m³ in policemen resp. 36.1 ± 31.6 ng/m³ in bus drivers in Sofia, 26.8 ± 39.8 ng/m³ for policemen in Kosice and 11.9 ± 11.2 ng/m³ for policemen in Prague), compared to the respective controls (24.9 ± 17.7 ng/m³ for controls in Sofia, 7.9 ± 3.8 ng/m³ for controls in Kosice and 6.2 ± 3.6 ng/m³ for controls in Prague).

We observed the following frequency of % AB.C. scored by conventional method: 2.60 ± 2.64 in exposed policemen and 2.14 ± 1.61 in controls in Kosice (p = n.s.); 2.33 ± 1.53 in exposed policemen and 1.94 ± 1.28 in controls in Prague (p = n.s.); 3.04 ± 1.64 in exposed policemen, respectively, 3.60 ± 1.63 in exposed bus drivers and 1.79 ± 0.77 in the control group in Sofia (p < 0.05, respectively, p < 0.05).

According to data from multiple regression analysis, and group comparison of smokers versus nonsmokers in Sofia also cigarette smoking (p = 0.055) and the age (p = 0.020) seem to play an important role within the aberrant cell formation in addition to the occupational c-PAHs exposure (p = 0.000). Smoking status was the modifying factor for % AB.C. in Kosice (p = 0.020) after multiple regression approach was employed.

In summary, we can say that subjects occupationally exposed to higher levels of c-PAHs in ambient air in Sofia are at greater genotoxic risk compared to those working indoors.     

Associations between common variation in the aromatase gene promoter region and testosterone concentrations in two young female populations

We recently reported association between a coding-region single nucleotide polymorphism (SNP50) in the aromatase gene that encodes a key enzyme in testosterone metabolism, with risk for the development of precocious pubarche and circulating testosterone concentrations in two independent female populations. We have now explored further association with variation in the promoter-region of the aromatase gene. We genotyped six promoter-region haplotype-tag SNPs in young women from Oxford, UK (n = 109), and in girls with precocious pubarche (n = 186) and controls (n = 71) from Barcelona, Spain. Aromatase distal promoter-region variation was associated with plasma testosterone concentrations in both Oxford (r² = 18.3%, p = 0.01) and Barcelona (r² = 8.5%, p = 0.03) females. These associations were independent of SNP50, but appeared to be dependent on different SNPs in Oxford (r² = 13.7%, p = 0.006 with SNPs 11 (p = 0.009), 28 (p = 0.02) and 39 (p = 0.06)) and Barcelona (r² = 5.9%, p = 0.002 with SNP43 (p = 0.002)) populations. Aromatase distal promoter-region variation was also associated with PCOS symptom score in Oxford women (r² = 14.5%, p = 0.048), but, unlike SNP50, was not associated with precocious pubarche risk in Barcelona girls. In conclusion, aromatase distal promoter-region variation appears to have functional consequences for plasma testosterone concentrations in females. The variable associations with androgen-related clinical features could possibly reflect the tissue-specific promoters of the aromatase gene.     

Attenuated feeding responses to circadian and palatability cues in mice lacking neuropeptide Y

Neuropeptide Y (NPY) is a potent orexigenic peptide that is implicated in the feeding response to a variety of stimuli. The current studies employed mice lacking NPY (Npy−/−) and their wild-type (Npy+/+) littermates to investigate the role of this peptide in the feeding response to circadian and palatability cues. To investigate the response to a circadian stimulus, we assessed food intake during the 4-h period following dark onset, a time of day characterized by maximal rates of food consumption. Compared to Npy+/+ controls, intake of Npy−/− mice was reduced by 33% during this period (0.6 ± 0.1 g versus 0.9 ± 0.1 g; p ≤ 0.05). In contrast, intake did not differ between genotypes when measured over a 24-h period (3.7 ± 0.2 g versus 3.5 ± 0.3 g; p = ns). Furthermore, reduced dark cycle 4 h food intake in Npy−/− mice was not evident after a 24-h fast (1.4 ± 0.1 g for both genotypes; p = ns), despite a pronounced delay in the initiation of feeding (636 ± 133 s versus 162 ± 29 s; p ≤ 0.05). To investigate the role of NPY in the feeding response to palatability cues, mice were presented with a highly palatable diet (HP) for 1 h each day (in addition to having ad libitum access to chow) for 18 days. Npy+/+ mice rapidly increased daily HP intake such that by the end of the first week, they derived a substantial fraction of daily energy from this source (41 ± 3%). By comparison, HP intake was markedly reduced in Npy−/− mice during the first week (24 ± 7% of daily energy intake, p ≤ 0.05 versus Npy+/+), although it eventually increased (by Day 9) to values comparable to those of Npy+/+ controls. These experiments suggest that NPY contributes to the mechanism whereby food intake increases in response to circadian and palatability cues and that mechanisms driving food intake in response to these stimuli differ from those activated by energy restriction.     

Attenuation of radiation-induced genomic instability by free radical scavengers and cellular proliferation.

To investigate the mechanisms of radiation-induced chromosomal instability, cells were irradiated in the presence of the free radical scavengers DMSO, glycerol, or cysteamine, in the presence of DMSO while frozen, or held in confluence arrest post-irradiation to permit cells to repair potentially lethal DNA damage. Clones derived from single progenitor cells surviving each treatment were then analyzed for the subsequent development of chromosomal instability. The presence of scavengers (+/- freezing) during irradiation, and the recovery from potentially lethal damage after irradiation led to an increase in cell survival that was accompanied by a decrease in the initial yield of chromosomal rearrangements. Furthermore, analysis of over 400 clones and 80,000 metaphases indicates that these same treatments reduced the incidence of instability at equitoxic doses when compared to controls irradiated in the absence of scavengers at ambient temperature. Results suggest that preventing reactive species from damaging DNA, promoting chemical repair of ionized DNA intermediates, or allowing enzymatic removal of genetic lesions, represent measures that reduce the total burden of DNA damage and reduce the subsequent onset of radiation-induced genomic instability.     

Chromosomal aberrations by fluorescence in situ hybridization (FISH)--Biomarker of exposure to carcinogenic PAHs

The fluorescence in situ hybridization (FISH) technique with whole chromosome painting for chromosomes #1 and #4 was used to study the impact of air pollution containing higher concentrations of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in three European cities, Prague (Czech Republic), Kosice (Slovakia) and Sofia (Bulgaria). In each site were followed an exposed group, who were police officers or bus drivers who work usually through busy streets for at least 8 h, and a reference group, who spent more than 90% of their daily time indoors.

In Prague, a significant increase was observed in percentage of aberrant cells (% AB.C.) in the police officers compared to the reference group (0.33 ± 0.25 versus 0.24 ± 0.18, p < 0.05). In Kosice, the exposed group differed from reference in the endpoints F_G/100 1.52 ± 1.18 versus 1.12 ± 1.30, p < 0.05; % AB.C. 0.30 ± 0.19 versus 0.21 ± 0.20, p < 0.05; t/1000 3.91 ± 3.18 versus 2.84 ± 3.10, p < 0.05. In Sofia were followed two exposed groups: police officers and bus drivers. All FISH endpoints were significantly higher in police officers compared to reference group (F_G/100 1.60 ± 0.99 versus 0.82 ± 0.79, p < 0.01; % AB.C. 0.25 ± 0.14 versus 0.13 ± 0.13, p < 0.01; t/1000 4.19 ± 2.65 versus 2.13 ± 2.05, p < 0.05; rcp 1.46 ± 1.07 versus 0.70 ± 0.76, p < 0.05). In bus drivers compared to reference there was an increase in % AB.C. (0.25 ± 0.18 versus 0.13 ± 0.13, p < 0.05).

This is the first study when FISH method was used to analyze the impact of environmental air pollution. According to the original hypothesis it is expected that the most important group of chemicals responsible for the biological activity of air pollution represent c-PAHs.     

Serum pancreatic lipase [EC 3.1.1.3] activity, serum lipid profile and peripheral blood dendritic cell populations in normolipidemic males with psoriasis

The purpose of the study was to explore serum pancreatic lipase activity and the serum lipid profile in relation to peripheral blood dendritic cell subsets and disease severity in males with psoriasis.

Material and methods

The study population consisted of 22 normolipidemic males with psoriasis and 12 aged-matched and body mass index (BMI)-matched healthy males. The percentages of peripheral blood dendritic cell (DC) subsets were evaluated using appropriate monoclonal antibodies and flow cytometry. The serum pancreatic lipase activity and the lipid profile were determined using standard enzymatic and colorimetric techniques.

Results

Pancreatic lipase activity was increased (p = 0.56421), high-density lipoprotein (HDL)-cholesterol concentration (p = 0.00584) was significantly decreased, triglyceride (p = 0.00766) and VLDL-cholesterol (p = 0.00765) levels were significantly increased in serum of psoriatic patients compared to controls. The serum pancreatic lipase activity showed significant correlation with serum triglyceride (r = 0.42; p = 0.04721) and serum VLDL-cholesterol levels (r = 0.42; p = 0.04721) in psoriatic individuals. In psoriatic patients the percentage of myeloid DCs was increased (p = 0.54932), the percentage of lymphoid DCs was decreased (p = 0.14210) and myeloid DC/lymphoid DC ratio was significantly increased (p = 0.03569) compared to healthy individuals.

Conclusion

The direct cause of the abnormal lipid profile in psoriasis and its relationship with the immune system disturbances remains unclear. The reciprocal relationship between serum pancreatic activity and serum triglyceride level appears to confirm the hypothesis about abnormal lipid metabolism in psoriasis.     

Consummatory behavior and metabolic indicators after central ghrelin injections in rats

Ghrelin, an endogenous ligand for the growth-hormone-secretagogue receptor, is a 28-amino acid peptide with a post-translational acyl modification necessary for its activity. It has central nervous system actions that affect appetite, body mass and energy balance. An intracerebroventricular (ICV) injection protocol of sub-nanomolar doses of ghrelin, known to alter the morphology of ACTH and GH producing pituicytes and plasma levels of these hormones, was used to provide an overview of metabolic changes linked to energy metabolism. Variables measured were: food intake (FI), water intake (WI), fecal mass, urine volume, body weight (BW), retroperitoneal (RP) and epididymal (EPI) white adipose tissue (WAT), and changes in serum leptin, insulin, triglycerides, cholesterol, and glucose. Five injections of rat ghrelin or PBS (n = 8 per group) were given ICV every 24 h (1 μg/5 μL PBS) to adult male rats. Ghrelin had a positive and cumulative effect on FI, WI and BW (p < 0.05), but not feces mass or urine volume (p > 0.05). Centrally applied ghrelin clearly increased RP WAT (by 235%, p < 0.001), EPI WAT (by 85%, p < 0.05) and serum insulin levels (by 43%, p < 0.05), and decreased serum leptin levels (by 77%, p < 0.05) without (p > 0.05) evoking changes in blood triglyceride cholesterol, or glucose levels.

These data and the available literature clearly document that exposure of the brain of normal rats, over time, to sub-nanomolar doses of ghrelin results in metabolic dysregulation culminating in increased body mass, consummatory behavior, and lipid stores as well as changes in blood leptin/insulin levels. Thus, modulation of central ghrelin receptors may represent a pharmacological approach for controlling multiple factors involved in energy balance and obesity.     

Abrogation of the effects of aphidicolin on NIH3T3 and V79 cells by caffeine

In this communication I show that caffeine (1,3,7-trimethylxanthine) stimulates [³H]thymidine incorporation in aphidicolin-treated V79 and NIH3T3 cells. Flow microfluorometric analysis showed that caffeine, partially or fully, abrogates the cell cycle progression block produced by aphidicolin. Increased cell growth is also observed in cultures treated with both aphidicolin and caffeine compared to cultures treated with aphidicolin only. Microscopic examination of V79 cultures treated with aphidicolin for 8 h showed a marked reduction in the freqeuncy of round mitotic cells, as is expected from a drug which inhibits progression through the cell cycle by inhibiting DNA replication; this effect of aphidicolin was also reduced by caffeine. Biochemical analysis showed that caffeine did not directly interfere with the inhibition of DNA polymerase-α by aphidicolin. Analysis of dNTP pools indicated that caffeine increased the level of dCTP in V79 cells. In aphidicolin-treated V79 cells, the increase in the dCTP level due to exogenous cytidine was almost completely blocked; caffeine also substantially overcame this effect of aphidicolin. These results indicate that caffeine produces its effects on aphidicolin-treated cells by altering the dCTP metabolism.     

Falls are associated with decreased renal function and insufficient calcitriol production by the kidney

In a double blind placebo controlled 3-year osteoporosis study in elderly women, we collected prospective data on falls. The study population comprised 489 normal elderly women aged 65–77 years randomized to four groups: placebo, calcitriol 0.25 mcg b.i.d., conjugated equine estrogens (0.625 mg/day) and calcitriol + estrogen. Falls occurred in 57% of all women.

Using a Poisson regression model, the placebo group with low GFR-creatinine clearance (CrCl < 60 ml/min) had 60% more falls compared to the group with CrCl ≥ 60 ml/min. Further sub group analyses showed that there is no increased risk of falls with CrCl 60–70, 70–80 and >90 ml/min. Calcitriol treatment significantly reduced the number of falls by 50% (OR = 0.5, 95% CI: 0.4–0.9, p = 0.010) compared to placebo in the low CrCl group.

The group with lower CrCl had lower calcium absorption (p < 0.001), lower serum 1,25-dihydroxyvitamin D (1,25(OH)₂D) (p < 0.001) and normal serum 25OHD suggesting that there is decreased conversion of 25OHD to 1,25(OH)₂D by the aging kidney. It is postulated that the decrease in falls on calcitriol therapy is related to an increase in serum 1,25(OH)₂D, upregulation of VDR and improvement in muscle strength although one cannot exclude an effect on the central nervous system.     

Short term effects of dexamethasone on hyaluronidase activity and sperm characteristics in rams

The aim of this study was to determine the effects of dexamethasone on sperm characteristics and hyaluronidase activity of serum and semen. In this investigation, 14 healthy Akkaraman rams, at the age of 2 years and weighing between 50–60 kg, were used. The rams were randomly divided into two groups. After the last administration of dexamethasone intramuscularly at a dose of 0.25 mg/kg, semen and blood samples were taken at different times. The results showed that the serum hyaluronidase activity was increased significantly (p < 0.001) in the treatment group when compared with the control group except for the 1st hour. There was a significant difference (p < 0.001, 0.01, 0.05) in the hyaluronidase activity of semen between the treatment group and the control group. Furthermore, there was a significant difference (p < 0.01) in sperm concentration between both groups at all the times except the 96th hour. There were statistically significant (p < 0.05) differences in semen volume between the treatment and control groups. There were also significant differences (p < 0.05) in sperm motility between the treatment and control groups except for the 72 and 96th hours.

These findings indicate that dexamethasone increases hyaluronidase activity of serum and semen, but it decreases sperm concentration, semen volume and sperm motility in rams. Therefore the use of these drugs in breeding rams during breeding season is not suitable.     

Caprine luteinizing hormone isoforms during the follicular phase and anestrus

The relative proportion of the circulating luteinizing hormone isoforms in goats during follicular phase (pre-ovulatory peak; F) and anestrus (A) was investigated. Estrus was synchronized in six goats with a prostaglandin analogue. After estrus was detected, blood samples were taken at 1 h intervals for 24 h. Four anestrous goats received 100 μg i.v. of GnRH and blood samples were collected every 15 min for 5 h. Samples with the greatest LH concentration in follicular phase and after GnRH administration (anestrus) were analyzed by chromatofocusing and eluted with a pH gradient from 10.5 to 3.5. For quantification purposes eluted LH was grouped into basic (pH ≥ 7.5), neutral (pH 7.4–6.5) and acidic isoforms (pH ≤ 6.4) as well as by pH unit. In both physiological conditions (PC), basic and acidic isoforms were greater than the neutral. With this grouping criteria, there was an interaction between PC and pH group, with the proportion of neutral isoforms being greater (p < 0.05) in A (12.0 ± 0.8%) as compared with F (5 ± 2%). Analysis by pH unit showed a very basic group of eluted isoforms (pH ≥ 10), which amounted to a percentage of 6.0 ± 0.4% of the total observed during A, and 3 ± 1% during F (p < 0.05). Predominant isoforms in A eluted in the pH range 9.99–9.0 (42 ± 3%) as compared to 7 ± 3% (p < 0.01) in that pH range in F. In contrast, the predominant isoforms in F eluted in the pH range 8.99–8.0, representing 55 ± 8%, while in A the proportion was 11 ± 2% (p < 0.01). Isoforms eluted at the pH range 7.9–7 represented a significantly greater proportion during A (5.0 ± 0.6%) as compared with F (3 ± 1%). This is the first report on goat LH circulating isoforms. During A the LH isoforms secreted by the pituitary are more basic than during F.     

The morphology of the medial gastrocnemius in typically developing children and children with spastic hemiplegic cerebral palsy

We collected 3D ultrasound images of the medial gastrocnemius muscle belly (MG) in 16 children with spastic hemiplegic cerebral palsy (SHCP) (mean age: 7.8 years; range: 4–12) and 15 typically-developing (TD) children (mean age: 9.5 years; range: 4–13). All children with SHCP had limited passive dorsiflexion range on the affected side with the knee extended (mean ± 1SD: −9.3° ± 11.8). Scans were taken of both legs with the ankle joint at its resting angle (RA) and at maximum passive dorsiflexion (MD), with the knee extended. RA and MD were more plantar flexed (p < 0.05) in children with SHCP than in TD children.

We measured the volumes and lengths of the MG bellies. We also measured the length of muscle fascicles in the mid-portion of the muscle belly and the angle that the fascicles made with the deep aponeurosis of the muscle. Volumes were normalised to the subject’s body mass; muscle lengths and fascicle lengths were normalised to the length of the fibula.

Normalised MG belly lengths in the paretic limb were shorter than the non-paretic side at MD (p = 0.0001) and RA (p = 0.0236). Normalised muscle lengths of the paretic limb were shorter than those in TD children at both angles (p = 0.0004; p = 0.0003). However, normalised fascicle lengths in the non-paretic and paretic limbs were similar to those measured in TD children (p > 0.05). When compared to the non-paretic limb, muscle volume was reduced in the paretic limb (p < 0.0001), by an average of 28%, and normalised muscle volume in the paretic limb was smaller than in the TD group (p < 0.0001).

The MG is short and small in the paretic limb of children with SHCP. The altered morphology is not due to a decrease in fascicle length. We suggest that MG deformity in SHCP is caused by lack of cross-sectional growth.     

Measuring pad-pad pinch strength in a non-human primate: Macaca fascicularis.

The primary purpose of this study was to establish a methodology for determining and perhaps predicting (via regression analysis of anthropometric measures) Macaca fascicularis isometric pinch strength for a specific task. The larger purpose of this work was to properly scale a pinching task for the monkeys in order to study dose-response relationships in a non-human primate model for carpal tunnel syndrome. Three female and one male macaque (n = 4) of varying size and age were trained to perform a left-handed pad–pad pinch. The task required 60° of wrist flexion at a static pinching distance of 3 cm between the thumb and fingers. Subjects were trained for a period of 20-weeks. After that time, an analysis of performance gradients found that they had each reached a plateau in their force output. Pinch strength for the four animals ranged from 29.4 to 59.8 N. Regression analysis revealed that body mass (kg) and wrist circumference (cm) were both predictive of pinch strength, exhibiting adjusted R² values of 0.93 (p = 0.024) and 0.96 (p = 0.015), respectively. Thus, the results suggest that maximal pinch strength could be acceptably estimated in future subjects using either the wrist circumference or the body mass measures, as both were strong predictors of pad–pad pinch strength.     

Ageing and dexamethasone associated sarcopenia: peculiarities of regeneration

The purpose of this study was to assess the development of ageing- and glucocorticoid-related sarcopenia on the level of myofibrillar apparatus, paying attention to the synthesis (SR) and degradation rate (DR) of contractile proteins, muscle strength, and daily motor activity. We also wanted to test the effect of ageing and dexamethasone (Dex) excess on the regeneration peculiarities of skeletal muscle autografts. Four and 30-month-old male rats of the Wistar strain were used. Ageing associated sarcopenia was calculated from gastrocnemius muscle relative mass decrease (from 5.6 ± 0.08 to 3.35 ± 0.04; p < 0.001). The SR of MyHC in old rats was 30% and actin 23% lower than in young rats. Dex treatment decreased SR of two main contractile proteins significantly in both age groups (p < 0.001) and increased DR during ageing from 2.11 ± 0.15 to 4.09 ± 0.29%/day (p < 0.001). Hindlimb grip strength in young rats was 5.90 ± 0.35 N/100 g bw and 2.64 ± 0.2 N/100 g bw (p < 0.001) in old rats.

Autografts of old rats have a higher content of adipose tissue 14.9 ± 1.1% in comparison with young rats 6.8 ± 0.51% (p < 0.001) and less muscle tissue 39.8 ± 2.6% and 48.3 ± 2.8%, respectively (p < 0.05).

Both, ageing and dex-caused sarcopenic muscles have diminished capacity for regeneration.     

RAPD and ISSR markers indicate diminished gene flow due to recent fragmentation of Polylepis australis woodlands in central Argentina

Two Polylepis australis BITT. populations differing in size were examined with the aim of determining future sampling strategies; assessing levels of genetic diversity and checking whether trees of different ages might vary in their genetic structure due to the effects of fragmentation. RADP and ISSR gave similar values of diversity. A re-sampling technique showed that for P. australis, 10 trees and 20 markers were enough to produce an unbiased estimator of heterozygosity. AMOVA suggested differences in allele frequencies between young and old trees in the small population (p = 0.052), but not in a large population (p = 0.864); suggesting that gene flow between the areas diminished in relatively recent times. This assumption is supported by the fact that allele frequencies among both woodlands were significantly different between the young (p < 0.0001), but not the older trees (p = 0.87).     

Biopsied and vitrified bovine embryos viability is improved by trans10, cis12 conjugated linoleic acid supplementation during in vitro embryo culture

Bovine embryos cultured in serum-containing media abnormally accumulate lipids in the cytoplasm. This is well known to contribute to their higher susceptibility to cryopreservation and biopsied embryos are even further susceptible. We aimed to improve in vitro produced (IVP) embryos resistance to micromanipulation and cryopreservation by supplementing serum-containing media with trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA). The effect of t10, c12 CLA on lipid deposition and embryonic development was also tested. After in vitro maturation and fertilization (IVF day = D0), zygotes were cultured on granulosa cells + M199 + 10% serum + 100 μM GSH supplemented with 100 μM of t10, c12 CLA (CLA group, n = 1394) or without supplementation (control group, n = 1431). Samples of D7/D8 embryos were observed under Nomarsky microscopy for lipid droplets evaluation while others were biopsied and vitrified (group B-Control, n = 24; group B-CLA, n = 23). Non-biopsied embryos were also frozen (group NB-Control, n = 49; group NB-CLA, n = 45). Biopsied cells were used for embryo sex determination. Postwarming embryo survival and viability were determined at 0 and 24 h of culture, respectively. Supplementation of t10, c12 CLA did not influence cleavage, embryo sex ratio, D7/D8 embryo rate or morphological quality. CLA embryos had higher number of small lipid droplets (P ≤ 0.003) and a smaller (P < 0.001) fat embryo index being leaner (P = 0.008) than control embryos. Embryo postwarming survival was higher in B-CLA than in B-control group (95.0 ± 7.0% versus 62.5 ± 7.9%; P < 0.001). After 24 h of culture, the viability (expansion rate) of biopsied embryos and nonbiopsied embryos, cultured with t10, c12 CLA was higher than control embryos (B-CLA = 64.6 ± 4.4% and B-control = 27.5 ± 2.5%, P = 0.01; NB-CLA = 86.0 ± 3.5% and NB-Control = 68.6 ± 7.0%, P = 0.05). Results showed that supplying t10, c12 CLA to serum-containing media decreases embryo cytoplasmic lipid deposition during in vitro culture and significantly improves resistance of IVP embryos to micromanipulation and cryopreservation.     

Effect of water renewal on dominance hierarchy of juvenile Nile tilapia

Nile tilapia social position (Oreochromis niloticus) can be mediated by multiple channels, including chemical communication. Absence of chemical cues in the environment prevents hierarchical settlement among pairs, and enhances time spent in confrontations. The aim of this study was to test the effect of continuously renewed water flow on the establishment of hierarchical dominance in Nile tilapia juveniles. In this condition, a high frequency of attacks and disruption on hierarchical stability were expected because chemical cues for hierarchy maintenance could be washed out. After 3 days in isolation, the fish were paired by standard size but not by sex, and submitted to two conditions: continuously renewed water flow (RENEWED, n = 7) and non-renewed water flow (NONRENEWED, n = 8). The paired fish were placed in an aquarium (40 cm × 30 cm × 40 cm) for 3 h; four 10-min sessions were video-recorded: the first, immediately after the fish were paired and the others 1, 2, and 3 h after pairing. Hierarchy was identified by a dominance index (DI = given attacks/received + given attacks) for each fish. The hierarchical stability was achieved by analyzing the difference between dominant DI and subordinate DI (DI-D). Hierarchy was established in both groups after second session because the DI was significantly higher for one fish of the pair. The frequency of attacks of the dominant fish in RENEWED and NONRENEWED conditions was similar in all observation sessions. The attack frequency by subordinate fish was also similar during the first three sessions (2-h pairing). However, the frequency of attacks by subordinate fish in the RENEWED condition was higher than in the NONRENEWED situation at the fourth observation session (means ± S.E.: RENEWED = 2.83 ± 0.94 × 10 min⁻¹ and NONRENEWED = 0.25 ± 0.16 × 10 min⁻¹; Mann–Whitney, p = 0.04). At this point, a significant reduction of the DI-D was observed (means ± S.E.: RENEWED = 0.70 ± 0.11 and NONRENEWED = 1,00 ± 0.002; Mann–Whitney, p = 0.04). The changes in DI-D were related to more frequent attacks by the subordinated fish in renewed water flow. According to our results, the unsteady agonistic interaction under renewed water flow leads to social instability. Thus, continuous water renewing can wash out relevant chemical substances and therefore disturb the dominance recognition by subordinate fish.     

Serum concentrations of Cu, Zn, Fe, Mo and Co in newborn lambs following systemic administration of Vitamin E and selenium to the pregnant ewes

This study was conducted to determine the effects of systemic administration of Vitamin E and selenium to pregnant ewes at the late stage of gestation on serum concentrations of Cu, Zn, Fe, Mo and Co in their offspring's. Pregnant Lori–Bakhtiari ewes (n = 14) were randomly assigned to receive Vitamin E and selenium (treatment group; n = 7) or distilled water (control group; n = 7), once 3 weeks and again 1 week before parturition. Blood samples were taken from jugular vein of lambs at parturition and once a week during the 4 week of age. Serum concentration of Fe, Cu, Zn, Mo and Co was determined by atomic absorption spectrophotometery.

At parturition, serum concentration of Cu, Zn, Mo and Co were identical in lambs of both groups, while the serum concentration of Fe (mean ± S.E.) was significantly higher in lambs of control (210.83 ± 9.05 μg/dl) than treatment group (140.71 ± 17.8 μg/dl).

From parturition to the forth weeks of age the serum concentrations of Fe and Cu were increased (P < 0.05) in lambs of treated group which was concomitant with a reduction in Zn concentration.

In conclusion, increase in serum concentrations of Cu and Fe during the first 4 weeks of age in lambs of ewes given vitamin E and selenium compound, could disturb the Zn:Cu and Zn:Fe ratios which in turn lead to zinc deficiency.     

Dydrogesterone in the reduction of recurrent spontaneous abortion

One hundred and eighty Women with a history of recurrent, unexplained spontaneous abortion (mean 3.5 abortions) were randomised to receive oral dydrogesterone (10 mg b.i.d.), intramuscular human chorionic gonadotrophin (hCG; 5000 IU every 4 days) or no additional treatment (controls). Treatment was started as soon as possible after confirmation of pregnancy and continued until the 12th gestational week. All women received standard supportive care. Abortions were significantly (p ≤ 0.05) less common in the dydrogesterone group (13.4%) than in the control group (29%); there were no statistically significant differences between the hCG group and the control group. There were no differences between the groups with respect to pregnancy complications or congenital abnormalities. In conclusion, hormonal support with dydrogesterone can increase the chances of a successful pregnancy in women with a history of recurrent spontaneous abortion.