An automated cell viability quantification method for low-resolution confocal images of closely packed cells based on a modified gradient flow tracking algorithm.

Fluorescent-based live/dead labelling combined with fluorescent microscopy is one of the widely used and reliable methods for assessment of cell viability. This method is, however, not quantitative. Many image-processing methods have been proposed for cell quantification in an image. Among all these methods, several of them are capable of quantifying the number of cells in high-resolution images with closely packed cells. However, no method has addressed the quantification of the number of cells in low-resolution images containing closely packed cells with variable sizes. This paper presents a novel method for automatic quantification of live/dead cells in 2D fluorescent low-resolution images containing closely packed cells with variable sizes using a mean shift-based gradient flow tracking. Accuracy and performance of the method was tested on growth plate confocal images. Experimental results show that our algorithm has a better performance in comparison to other methods used in similar detection conditions.

Effect of the slow (K) or rapid (k+) feathering gene on body and feather growth and fatness according to ambient temperature in a Leghorn x brown egg type cross.

Chicks of both sexes issued from the cross of heterozygous K/k+ cocks for the slow-feathering sex linked K allele with k+ (rapid feathering) hens, were compared from the age of 4 to 10 weeks at two ambient temperatures. In individual cages, 30 male chicks of each genotype (K/k+ and k+/k+) were raised at 21 degrees C, and 60 others, distributed in the same way, were raised at 31 degrees C. 71 K/W females and 69 k+/W females were raised in a floor pen at 31 degrees C till 10 weeks of age. In the males, the body weight, feed consumption and feed efficiency at different ages were influenced only by temperature (lower growth rate and feed intake at 31 degrees C); no significant effects of the genotype at locus K nor genotype x temperature interaction were observed. In females, all at 31 degrees C, the genotype (K/W or k+/W) had no significant effect on growth rate. Plumage weight and weight of abdominal fat (absolute or related to body weight) were measured on half of the males of each group in individual cages, at 10 weeks of age. Moreover, on 36 males and 48 females of the two genotypes, in a group battery at 31 degrees C, the absolute and relative weight of plumage were measured on a sample every two weeks between 4 and 10 weeks. In the first case, no significant effect of genotype appeared. In the second case, an interaction between age and genotype was suggested from plumage weight: its growth, especially in male chicks, appears to be temporarily and unexpectedly faster from 4 to 6 weeks of age for the K/k+ and K/W genotypes.

Unmet needs in hypertension: challenges and opportunities.

On the verge of the new millennium, hypertension remains an area of significant unmet medical need. Although progress has been made in the awareness, treatment, and control of hypertension during the last half of the 20th century, recent trends suggest that progress has stalled. Unfavorable trends in awareness and control have been noted by researchers at the Mayo Clinic, in a community that is socioeconomically prosperous with easy access to primary and tertiary medical care. Evidence suggests that a renewed focus on systolic blood pressure and on efforts to maintain compliance will result in better outcomes in populations at risk. Hiatt and Goldman have presented the case for "making medicine more scientific"--that is, understanding how the application of medical care advances can best be applied to benefit population health status. This is the challenge that hypertension control presents in world populations. Aging societies make this challenge an urgent concern.

Heterosis in egg-laying lines under divergent selection for residual feed consumption.

Two lines selected since 1976 for high (R+) or low (R-) residual feed consumption (RFC) from a common genetic base were compared with one another and with their F1 reciprocal crosses for traits of egg production and quality, for morphological traits, body weights, and feed consumption. Heterosis was 11, -2.5, 8, and 2%, respectively, for egg number, age at first egg, egg laying rate, and egg weight, with marked differences between reciprocal crosses for all those traits but egg number. Heterosis for wattle length and shank length was 3.8 and 1.3%, respectively, essentially because R+ x R- crossbreds, with larger mean values, resembled the R+ line for those traits, which may therefore be associated with the presence of genes linked to the Z chromosomes. On the other hand, heterosis for RFC (-3.6%) originated from similar crossbred advantage in both reciprocal crosses, thereby suggesting that RFC is not determined by sex-linked genes.

Cytogenetic study of early chicken embryos: effect of naked neck gene and high ambient temperature.

High ambient temperature (> 30 C) decreases fertility of breeder hens, but this effect has been shown to be greatly reduced in females carrying the naked neck gene (Na). Sixty-four females each of the three different genotypes, Na/Na (homozygous naked neck), Na/na+ (heterozygous), and na+/na+ (normally feathered), were equally distributed in two climate control rooms with individual cages, at a constant temperature of either 22 C or 31 C. Five hundred and seventy-six embryos were examined after 16 to 18 h of incubation for karyological analyses. Abnormalities consisted of diploid-haploid and diploid-triploid chimeras. The frequency of chimeric embryos was significantly affected by dam genotype. Naked neck females showed a much lower proportion of abnormal embryos than normally feathered females whatever the temperature. The highest proportion of chimeras was observed for the na+/na+ dams maintained at 31 C. However, the effect of temperature was not generally significant.

Comparison of ultradian and circadian oscillations of carbon dioxide production by various endotherms.

Carbon dioxide emission (VCO2) was computed every 20 min from continuous CO2 concentration recordings taken during 3-30 consecutive days, in strictly controlled environmental conditions, in 54 OF1 mice, 99 Japanese quail, 66 Sprague-Dawley rats, 50 Hartley guinea pigs, 7 chicks, for 7-15 days on 2 Cynomolgus monkeys, and for 24 h on 7 premature infants. This VCO2 shows circadian and ultradian oscillations that were analyzed for frequencies and amplitudes in light-dark 12-h alternation (LD 12:12), continuous light (LL), and continuous dark (DD). Circadians were not always identified or were often masked in LL or DD (mostly in guinea pigs, quail, and rats), while ultradians (tau > or = 40 min) were found in all species, at every time, and in all light regimens. Analysis of variance and chi 2 show significant (P < 0.001) interspecies differences for ultradian (1.07 < tau < 1.40 h) intervals and for circadian and ultradian VCO2 amplitudes. Relationships between ultradian and circadian VCO2 oscillations differ according to the species, ultradians appearing as an entity characteristic for each endotherm species.

Heterosis in normal versus dwarf laying hens.

The effect of genotype at the sex-linked dwarf locus on heterosis in crosses between a White Leghorn and a brown egg line for body weight, egg production, and related traits was studied. Heterozygous Dw/dw males were used to produce normal and dwarf pullets in each of the pure lines and their reciprocal crosses (eight genotype-line combinations). There were 54 pullets per combination. Line differences were significant for shank length, body weights at 8, 17, and 52 wk, age at first egg, egg number, clutch length, rate of lay, and egg weight. Heterosis was observed for all of these traits. Body weight as a covariate was not important in analyses of egg number, clutch length, and egg weight. The egg production reduction associated with the dw gene in pure lines was smaller in F1 hens. This discovery may be adequate to warrant use of dwarf crossbred hens for egg production.

Effects of sex-linked imperfect albinism in the chicken (sal-c) on plasma luteinising hormone concentrations and early egg production.

1. When measured before and after the onset of darkness, plasma LH concentrations in 40-day-old sex-linked albino pullets (sal-c) were slightly lower than those of nonalbinos (s+). 2. This finding prompted an experiment in which plasma LH concentrations were measured between 12 and 33 weeks of age when daylength was increased at 15 or 21 weeks. Egg production of the early and late maturing albino hens was measured. 3. Plasma LH concentrations overall and at 17 weeks were lower for albinos than for nonalbinos. In the early maturing group egg production of albinos was higher than that of nonalbinos. 4. Results suggest that increased egg production of albinos is not the direct result differences in plasma LH concentrations but may be a consequence of differences in the control of LH secretion.

Research note: the S(al)-c mutation in the chicken is not linked to Rous-associated virus-2 related DNA sequences.

A male chicken heterozygous for a gene for sex-linked imperfect albinism (s(al)-c) was used to produce offspring in five dam families. The DNA from 23 offspring (22 females and 1 male) was cut with the restriction enzyme Sac1 and hybridized with a Rous-Associated Virus-2 probe to test for linkage between the gene and endogenous viral (ev) genes. In a second experiment, 15 albino and 15 nonalbino females resulting from 4 males mated to 11 females were studied with the enzymes Sac1 and BamHI. In the first experiment, 10 different ev genes were seen. These, plus seven additional bands were seen in the second experiment. The gene s(al)-c segregated independently of all ev genes, suggesting that it could be used for autosexing of chickens or in layer flocks without causing inherent problems previously associated with K, the gene for slow feathering, caused by essentially complete concordance between K and ev21.

Effects of light on melatonin and two enzymes leading to its production in albino (s(al-c)) and nonalbino chickens.

A gene for sex-linked imperfect albinism in the chicken (s(al)) has been associated with increased egg production with an implication that environmental light may play a role. In this study, levels of melatonin and hydroxyindole O-methyltransferase (HIOMT) and N-acetyltransferase (NAT), two enzymes leading to melatonin production, were studied in young albino and nonalbino chickens in relation to the daily light cycle, and after 19 days of constant light or dark. Differences between genotypes were found in the levels of HIOMT activity in the pineals and retinas of birds kept in constant light for 19 days. Other measurements were not significantly different. This study would appear to show that the visual system of imperfect albino chickens reacts differently to light than that of nonalbinos, but not with changes in the daily cycle of plasma melatonin or in NAT activity, which is the enzyme primarily responsible for the control of the melatonin level in the body.

Direct and correlated responses to divergent selection for residual food intake in Rhode Island Red laying hens.

1. Divergent selection was undertaken in a Rhode Island Red population for residual food intake, measured in males and females, using mass selection. 2. In the absence of a control line, selection response during 14 generations was estimated by the within-year divergence between lines. 3. The direct response in residual food intake was found to be significant in both sexes, the divergence reaching almost three phenotypic standard deviations in each sex. 4. Significant correlated responses were obtained for food efficiency; it was improved in the low residual food intake line. Shank length, wattle length and rectal and comb temperature showed higher values in the high line, suggesting an increased heat production or dissipation. Inconsistent changes were observed for other egg production traits.

Differential environmental effects on lesions, early growth, and mortality of imperfect albino (Sal-c) chicks.

A series of experiments investigated early pleiotropic effects of a gene for imperfect albinism (s(al-c) in a population of chickens at Jouy-en-Josas, France. An elevated incidence of lesions of the navel, hocks, and nares typical of imperfect albinos were seen on these chicks, confirming their existence in this population. Variations in hatching environment and the amount of light in the hatcher both implicated in contributing to the occurrence of lesions of the hocks and nares, but not to those of the naval. In two experiments, using batteries and cages, early growth was reduced among albinos and early mortality was increased. Significant genotype by environment interactions for weight at 4 days and growth to this time, but not thereafter, suggested that the effect is restricted to this time. Unfavorable environments were most deleterious to albinos. In a third experiment, conducted in floor pens, the gene had no effect on either early growth or mortality. There was no effect of the gene on the lesions, early growth, or mortality when carried by heterozygous males (s+/s(al-c)).

Transfer of genetic epilepsy by embryonic brain grafts in the chicken.

In the Fayoumi chicken, a spontaneous recessive autosomal mutation (F.Epi) is responsible for high susceptibility to seizures that are especially inducible by intermittent light stimulation. Substitution of defined areas of the encephalic neuroepithelium in normal chicken embryos at 2 days of incubation by their counterparts from homozygous F.Epi embryos generates the epileptic phenotype in the chimeras. It was found that grafting primordia of both prosencephalon and mesencephalon of homozygous F.Epi birds is necessary and sufficient for transfer of the full disease. When grafted alone, the homozygous F.Epi prosencephalon, although showing the typical epileptic interictal electroencephalogram, does not allow the complete epileptic seizures to occur in the hosts. Grafts of mesencephalon and/or rhombencephalon modify neither the behavior nor the electroencephalographic pattern of the recipient chickens. Cooperation of forebrain and midbrain activities is therefore required to yield epileptic seizures in this model.

Effects of dietary T3 on growth parameters and hormone levels in normal and sex-linked dwarf chickens.

Tri-iodothyronine (T3) has been administered in the diet, from day of hatch until 8 weeks of age, to sex-linked dwarf and normal chicks of both sexes from a brown-egg slow-growing strain. Feed was supplemented with either 0.1 ppm or 0.5 ppm T3. A significant genotype by treatment interaction was observed on body weight: the effect of T3 in males was significantly positive for dwarfs and null for normals, the effect in females was null for dwarfs and significantly negative for normals. Feed efficiency was rather decreased by the treatment in both genotypes. Abdominal fatness was decreased in a dose-dependent manner in both genotypes, while rectal temperature was raised in dwarf chicks only. Plasma T3 was raised to normal levels in dwarfs receiving 0.1 ppm exogenous T3, while the 0.5 ppm dose yielded hyperthyroid levels. Plasma GH levels were decreased in a dose-dependent manner by the T3 treatment, the effect being larger in dwarfs. Surprisingly, plasma IGF-I was unchanged in spite of the GH decrease, whatever the genotype or the sex. It was concluded that exogenous T3 alone can have a stimulatory effect on growth in dwarfs but can not fully restore a normal growth rate. Both T3 and IGF-I are important for a normal growth and the relationship between T3 and IGF-I production should be further investigated in order to better understand the physiological modifications due to the sex-linked dwarf gene.

Ultradian and circadian compartmentalization of respiratory and metabolic exchanges in small laboratory vertebrates.

Carbon dioxide emission (VCO2) taken as an index of respiratory and metabolic exchanges, was continuously recorded during 4-30 consecutive days in 100 quail, 87 chicks, 347 rats, 665 mice and 70 guinea-pigs which were under controlled environmental parameters. Harmonic analysis, fast Fourier transform, chi-square periodograms, peak and trough intervals were computed with VCO2 values obtained with CO2 concentrations sampled every 20 min on the CO2 recordings. In LD 12:12 alternation, circadian rhythms were observed in all quail, chicks, rats and mice, but only in 80% of the guinea-pigs. Ultradian VCO2 rhythms, with periods which show statistically significant interspecies differences, were assessed. For each of the 5 species these computed periods, which were the same in LL and DD, were: 1.17 h for quail and chickens, 1.25 h for rats, 1.50 h for mice and 1.0 h for guinea-pigs. In LD 12:12 these periods were different during L and D in quail, chicks, rats and mice, but not in guinea-pigs. The amplitudes of these ultradian variations were, according to the species, 10-20% of their mean VCO2 levels. These ultradian rhythms persist in the absence (or masking) of circadian rhythms, e.g. in LD 12:12 in 20% of guinea-pigs and in LL in 87% of Japanese quail and in 23% of Sprague-Dawley rats. Moreover, these ultradian rhythms persist during starvation, locomotor activity restraint and ageing. These ultradian VCO2 cycles which are related to rest-activity variations appear to be basic physiological rhythms with a genetic origin.

Increased in-vivo insulin sensitivity but normal liver insulin receptor kinase activity in dwarf chickens.

The effects of the recessive and sex-linked dw gene on insulin sensitivity and liver insulin receptors were compared in normal (Dw-dw) and dwarf (dw-dw) brother or half-brother chickens. At 3.5 weeks of age, following an overnight fast, exogenous insulin (0-6.9 nmol/kg body weight) was slightly but significantly more hypoglycaemic in dwarf chickens. At 4 weeks of age, following an oral glucose load (2 g/kg), glucose tolerance was the same in both genotypes, whereas plasma insulin levels were greatly decreased in dwarf chickens. At 5 weeks of age, plasma concentrations of glucose and insulin were the same in both genotypes in the fasting state and decreased in the fed state in dwarf chickens. In liver membranes prepared from fasted chickens, insulin binding was increased in dwarf chickens, while the affinity of insulin receptors and the insulin-degrading activity of the membranes were the same in both genotypes. Following solubilization with Triton X-100, liver receptors were successively purified on lentil then wheat germ lectins. Autophosphorylation of the beta-subunit did not differ between either the genotype or the nutritional (fed or fasted) state. In the basal state (in the absence of insulin) the tyrosine kinase activity of the receptor towards artificial substrate poly(Glu, Tyr)4:1 was significantly decreased in dwarf chickens by fasting. However, the change in tyrosine kinase activity of the receptor in response to insulin was similar, irrespective of the genotype and the nutritional state.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of the sex-linked dwarfing gene (dw) on the contribution of dietary lipid to yolk lipid synthesis.

1. The efficiency and time course of dietary fatty acid incorporation into lipids of egg yolk and abdominal adipose tissue was compared in "White Leghorn", normal (Dw) and dwarf (dw) laying hens at 56 weeks of age, using 14C labelled linoleic acid. 2. The sex-linked dwarfing gene, dw, was shown to reduce not only body weight and abdominal fat pad deposition, but also yolk production and the average clutch size. 3. Higher peak incorporation and total recovery of the linoleic acid radioactivity into yolk lipids, but lower label recovery into adipose tissue triglycerides were found in dwarf hens. 4. The higher esterification of the dietary linoleic acid in its native form into dwarf yolk triglycerides indicates that dwarf hens use more dietary lipids to synthesise yolk lipids but these results also suggest that the dw allele might reduce the lipogenic capacities of the liver and adipose tissue in laying hens.

Influence of the sex-linked dwarfing gene (dw) on the lipid composition of plasma, egg yolk and abdominal fat pad in White Leghorn laying hens: effect of dietary fat.

The levels and fatty acid composition of lipids were determined in very low density lipoproteins (VLDL, d less than 1.006), yolk and abdominal adipose tissue of normal (Dw) and sex-linked dwarf (dw) White Leghorn laying hens. Effects of adding 4% tallow to the diet were also examined. In 40-wk-old hens, neither plasma lipids (triglycerides, phospholipids and cholesterol), VLDL levels, nor the chemical composition of VLDL was altered by the dw gene or dietary fat. Dwarfism reduced egg and yolk weights. Though the yolk lipid content was similar in normal and dwarf hens, yolk from dwarfs had slightly more phospholipids and less triglycerides than yolk from normal hens. Higher linoleic acid [18:2(n-6)] and lower oleic acid [18:1(n-9)] levels were observed in triglycerides of VLDL, yolk and adipose tissue from dwarf hens. In addition, the dietary fatty acid pattern had a greater influence on the fatty acid composition of the yolk lipid major precursors (VLDL triglycerides) in dwarf laying hens than in normal hens. These results suggest that the dwarfing gene might reduce the hepatic de novo fatty acid synthesis and/or dwarf hens might incorporate more dietary lipids into yolk than do normal hens.

De novo lipogenesis and lipolysis activities in normal (Dw) and dwarf (dw) White Leghorn laying hens.

1. In vitro activities of glucose oxidation, de novo lipogenesis and lipolysis were compared in normal (Dw) and dwarf (dw) laying hens. 2. Dwarfism reduced the hepatic glucose oxidation while de novo lipogenesis was not altered. As liver weight was depressed, total liver lipogenesis capacity was probably reduced by dwarfism. 3. As compared to normal hens, de novo lipogenesis and basal or stimulated lipolysis were lower in dwarf adipose tissue while its lipid content was enhanced in dwarfs. 4. Results suggest that in laying hens dwarfism reduces the adipose tissue lipid mobilization but probably also the liver de novo lipogenesis.