Desempenho, composição da cama, das tíbias, do fígado e das excretas de frangos de corte alimentados com rações contendo fitase e baixos níveis de fósforo

Um experimento foi realizado para avaliar o desempenho e a composição da cama, das tíbias, do fígado e das excretas de frangos de corte em diversas fases utilizando-se diferentes níveis de fósforo disponível (Pd) e da enzima fitase. Foram utilizados 1.200 pintos machos de 1 dia de idade, da marca comercial Cobb, distribuídos em delineamento inteiramente casualizado, em esquema fatorial 4 × 3, composto de quatro níveis de fósforo disponível e três níveis de fitase: 0, 500 e 1.000 ftu/kg de ração, cada um com quatro repetições de 25 aves. Os níveis de fósforo disponível foram obtidos considerando quatro disponibilidades do fósforo total (Pt) do milho e do farelo de soja (34, 56, 78 e 100% de disponibilidade). Foram avaliados os índices de desempenho no final de cada fase de criação e aos 42 dias de idade. Os parâmetros de desempenho foram afetados pela redução dos níveis de fósforo disponível das rações e a redução desse elemento para níveis inferiores a 19% piorou o desempenho das aves, enquanto a utilização de fitase no nível de Pd reduzido em 39% melhorou o desempenho. Nas fases de criação, a redução do teor de fósforo disponível com a utilização da enzima fitase influencia o desempenho e as características da cama e dos ossos.

Envolvimento da proteína, carboidrato, lipídio e selênio sobre as alterações metabólicas e bioquímicas em frangos submetidos ao calor

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effects of different mating scenarios on embryo viability in brown trout.

Mating with attractive or dominant males is often predicted to offer indirect genetic benefits to females, but it is still largely unclear how important such non-random mating can be with regard to embryo viability. We sampled a natural population of adult migratory brown trout (Salmo trutta), bred them in vitro in a half-sib breeding design to separate genetic from maternal environmental effects, raised 2098 embryos singly until hatching, and exposed them experimentally to different levels of pathogen stress at a late embryonic stage. We found that the embryos' tolerance to the induced pathogen stress was linked to the major histocompatibility complex (MHC) of their parents, i.e. certain MHC genotypes appeared to provide better protection against infection than others. We also found significant additive genetic variance for stress tolerance. Melanin-based dark skin patterns revealed males with 'good genes', i.e. embryos fathered by dark coloured males had a high tolerance to infection. Mating with large and dominant males would, however, not improve embryo viability when compared to random mating. We used simulations to provide estimates of how mate choice based on MHC or melanin-based skin patterns would influence embryos' tolerance to the experimentally induced pathogen stress.

Viability and Management of an Endangered Capercaillie (Tetrao urogallus) Metapopulation in the Jura Mountains, Western Switzerland

The populations of Capercaillie (Tetrao urogallus), the largest European grouse, have seriously declined during the last century over most of their distribution in western and central Europe. In the Jura mountains, the relict population is now isolated and critically endangered (about 500 breeding adults). We developed a simulation software (TetrasPool) that accounts for age and spatial structure as well as stochastic processes, to perform a viability analysis and explore management scenarios for this population, capitalizing on a 24 years-long series of field data. Simulations predict a marked decline and a significant extinction risk over the next century, largely due to environmental and demographic stochasticity (average values of life-history parameters would otherwise allow stability). Variances among scenarios mainly stem from uncertainties about the shape and intensity of density dependence. Uncertainty analyses suggest to focus conservation efforts on enhancing, not only adult survival (as often advocated for long-lived species), but also recruitment. The juvenile stage matters when local populations undergo extinctions, because it ensures connectivity and recolonization. Besides limiting human perturbations, a silvicultural strategy aimed at opening forest structure should improve the quality and surface of available patches, independent of their size and localization. Such measures are to be taken urgently, if the population is to be saved.

Genetic and maternal contributions to offspring viability under different environmental conditions in various salmonids

Summary : Due to anthropogenic impacts and natural fluctuations, fish usually have to cope with constantly changing and often hostile environments. Whereas adult fish have various possibilities to counteract unfavourable environmental conditions, embryos have much fewer options. Besides by their developing immune system, they are protected by the egg envelopes and several immune substances provided by their mothers. In addition to this, they may also adjust their hatching timing in reaction to various risks. However, individuals may vary in their defensive potential. This variation may be either based on their genetics and/or on differential maternal investments and may be dependent on the experienced stress. Nevertheless, in fish, the impact of such parental contributions on embryo and/or juvenile viability is still poorly investigated. The main objective of this thesis was to investigate the importance of paternal (i.e. genetic) and maternal (i.e. genetic + egg investment) contributions to offspring viability under different environmental conditions and at different life stages. In order to investigate this, we used gametes of various salmonids for in vitro fertilisation experiments based on full-factorial breeding designs. The individual studies are summarised in the following chapters: In the first chapter, we tested the effectiveness of the embryonic immune system in Lake whitefish (Coregonus palaea). Namely, we investigated paternal and maternal contributions to the embryos' tolerance to different kinds of pathogen exposure. Additionally, we tested whether an early sub-léthal exposure has a positive or a negative effect on an embryo's susceptibility to later pathogen exposures with the same pathogen. We found that pre-challenged embryos were more susceptible to future challenges. Moreover, pathogen susceptibility was dependent on maternal investments and/or the embryos' own genetics, depending on the challenge level. Chapter 2 summarises a similar study with brown trout (Salmo trutta). In addition to the previously described investigations, we analysed if genetic effects on offspring viability are mediated either by parental MHC genotypes or relatedness based on neutral microsatellite markers, and we tested if males signal their genetic quality either by their body size or their melanin-based skin colouration. We found that embryo survival was lower at higher stress levels and dependent on the embryos' genetics. Addirionally, parents with similar and/or, very common MHC genotypes had higher offspring viabilities. Finally, darker males produced more viable offspring. In the first two chapters we investigated the embryos' defensive potential based on their immune system, i.e. their pathogen tolerance. In chapter 3 we investigate whether hatching timing of Lake whitefìsh (C. palaea) is dependent on parental contributions and/or on pathogen pressure, and whether there are parental-environmental interactions. We found that whitefish embryos hatch earlier under increasing pathogen pressure. Moreover, hatching timing was affected by embryo genetics and/or maternally provided resources, but the magnitude of the effect was dependent on the pathogen. pressure. We also found a significant paternal-environmental interaction, indicating that the hatching efficiency of a certain sib group is dependent on the pathogen environment. Chapter 4 describes an analogous study with brown trout (S. trutta), with similar findings. In the former chapters, we only looked at offspring performance during the embryonic period, and only under semi-natural conditions. In chapter 5 we now test the performance and viability of embryonic and juvenile brown trout (S. trutta) under natural conditions. To measure embryo viability, we put them in brood boxes, buried them in the gravel of a natural river, and analysed survival after several months. To investigate juvenile survival and performance, wé reared embryos under different stress levels in the laboratory and subsequently released the resulting hatchlings in to a closed river section. Juvenile size and survival was then determined one year later. Additionally, we investigated if sires differ in their genetic quality, determined by embryo and juvenile survival as well as juvenile size, and if they signal their quality by either body size or melanin-based body darkness. We found hat juvenile size was dependent on genetic effects and on maternal investment, whereas this was neither the case for embryo nor for juvenile survival. Additionally, we found that offspring of darker males grew larger, and larger juveniles had also an increased survival. Finally, we found acarry-over effect of the early non-lethal challenge: exposing embryos to higher stress levels resulted in smaller juveniles. To evaluate the long-term performance of differently treated groups, mark-recapture studies are inevitable. For this purpose, effective mass-marking techniques are essential. In chapter 6 we tested the suitability of the fluorescent pigment spray marking method for the mass marking of European graylings (Thymallus thymallus), with very promising results. Our in vitro fertilisation studies on whitefish may reveal new insights on potential genetic benefits of mate choice, but the mating system of whitefish under natural conditions is still poorly investigated. In order to study this, we installed underwater cameras at the spawning place of a Coregonus suidteri population, recorded the whole mating period and subsequently analysed the recordings. Confirmations of previous findings as well as exciting new observations are listed and discussed in chapter 7. Dus aux impacts anthropogéniques et aux fluctuations naturelles, les poissons doivent faire face à des environnements en perpétuel changement. Ces changements font que les poissons doivent s'adapter à de nouvelles situations, souvent hostiles pour eux. Les adultes ont différentes possibilités d'échapper à un environnement peu favorable, ce n'est par contre pas le cas des embryons. Les embryons sont protégés d'une part par leur système immunitaire en développement, d'autre part, par la coquille de l'eeuf et différentes substances immunitaires fournies par leur mère. De plus, ils sont capables d'influencer leur propre date d'éclosion en réponse à différents facteurs de stress. Malgré tout, les individus varient dans leur capacité à se défendre. Cette variation peut être basé sur des facteurs génétiques et/ou sur des facteurs maternels, et est dépendante du stress subi. Néanmoins, chez les poissons, l'impact de telles contributions parentales sur la survie d'embryons et/ou juvéniles est peu étudié. L'objectif principal de cette thèse a été d'approfondir les connaissances sur l'importance de la contribution paternelle (c.a.d. génétique) et maternelle (c.a.d. génétique + investissement dans l'oeuf) sur la survie des jeunes dans différentes conditions expérimentales et stades de vie. Pour faire ces analyses, nous avons utilisé des gamètes de divers salmonidés issus de croisements 'full-factorial'. Les différentes expériences sont résumées dans les chapitres suivants: Dans le premier chapitre, nous avons testé l'efficacité du système immunitaire des embryons chez les corégones (Coregonus palea). Plus précisément nous avons étudié la contribution paternelle et maternelle à la tolérance des embryons à différents niveaux de stress pathogène. Nous avons aussi testé, si une première exposition non létale à un pathogène avait un effet positif ou négatif sur la susceptibilité d'un embryon a une deuxième exposition au même pathogène. Nous avons trouvé que des embryons qui avaient été exposés une première fois étaient plus sensibles au pathogène par la suite. Mais aussi que la sensibilité au pathogène était dépendante de l'investissement de la mère et/ou des gènes de l'embryon, dépendamment du niveau de stress. Le deuxième chapitre résume une étude similaire avec des truites (Salmo truffa). Nous avons examiné, si la survie des jeunes variait sous différentes intensités de stress, et si la variance observée était due aux gènes des parents. Nous avons aussi analysé si les effets génétiques sur la survie des juvéniles étaient dus au MHC (Major Histocompatibility Complex) ou au degré de parenté des parents. De plus, nous avons analysé si les mâles signalaient leur qualité génétique par la taille du corps ou par leur coloration noire, due à la mélanine. On a trouvé que la survie des embryons était plus basse quand le niveau de stress était plus haut mais que la variation restait dépendante de la génétique des embryons. De plus, les parents avec des MHC similaires et/ou communs avaient des embryons avec une meilleure survie. Par contre, des parents avec un degré de parenté plus haut produisent des embryons avec une survie plus mauvaise. Finalement nous avons montré que les mâles plus foncés ont des embryons qui survivent mieux, mais que la taille des mâles n'a pas d'influence sur la survie de ces mêmes embryons. Dans les deux premiers chapitres, nous avons étudié le potentiel de défense des embryons basé sur leur système immunitaire, c.a.d. leur tolérance aux pathogènes. Dans le troisième chapitre, nous nous intéressons à la date d'éclosion des corégones (C. palea), pour voir si elle est influencée par les parents ou par la pression des pathogènes, et si il y a une interaction entre ces deux facteurs. Nous avons trouvé que les jeunes naissent plus rapidement lorsque la pression en pathogènes augmente. La date d'éclosion est influencée par la génétique des embryons et/ou l'investissement des parents, mais c'est la magnitude des effets qui est dépendante de la pression du pathogène. Nous avons aussi trouvé une interaction entre l'effet paternel et l'environnement, ce qui indique que la rapidité d'éclosion de certains croisements est dépendante des pathogènes dans l'environnement. Le chapitre 4 décrit une étude analogue avec de truites (S. truffa), avec des résultats sitzimilaires. Dans les précédents chapitres nous nous sommes uniquement concentrés sur les performances des jeunes durant leur stade embryonnaire, et seulement dans des conditions semi naturelles. Dans le chapitre 5 nous testons la performance et la viabilité des embryons et de juvéniles de truites (S. truffa) dans des conditions naturelles. Nous avons trouvé que la taille des juvéniles était dépendante d'effets génétiques et de l'investissement maternel, mais ceci n'était ni les cas pour les survie des embryons et des juvéniles. De plus, nous avons trouvé que les jeunes des mâles plus foncés devenaient plus grands et que les grands ont un meilleur taux de survie. Finalement nous avons trouvé un 'carry-over effect' d'une première exposition non létale à un pathogène: exposer des embryons à des plus hauts niveaux de stress donnait des juvéniles plus petits. Pour évaluer la performance à long terme de groupes traités dé manières différentes, une méthode de marquage-recapture est inévitable. Pour cette raison, des techniques de marquage en masse sont nécessaires. Dans le chapitre 6, nous avons testé l'efficacité de la technique `fluorescent pigment spray marking' pour le marquage en masse de l'Ombre commun (Thymallus thymallus), avec des résultats très prometteurs. Les études de fertilisations in vitro avec les corégones nous donnent une idée du potentiel bénéfice génétique que représente la sélection d'un bon partenaire, même si le système d'accouplement des corégones en milieu naturel reste peu connu. Pour combler cette lacune, nous avons installé des caméras sous-marines autour de la frayère d'une population de corégones (C. suidteri), nous avons enregistré toute la période de reproduction et nous avons analysé les données par la suite. Ainsi, nous avons été capables de confirmer bien des résultats trouvés précédemment, mais aussi de faire de nouvelles observations. Ces résultats sont reportés dans le septième chapitre, où elles sont comparées avec des observations antérieures.

Influence of Steroidal Anti-Inflammatory Drugs on Viability and Fertility of Equine Semen

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison of Two Staining Methods for Pollen Viability Studies in Sugarcane

The present work aimed to compare two staining methods for pollen viability evaluation in sugarcane. Pollen from four sugarcane genotypes were collected at three different times (6.00, 8.00 and 9.00 a.m.) and tested for viability using two staining methods (iodine and lactophenol blue). Three anthers, of each genotype were crushed in a glass slide with a drop of the respective stain (iodine 0.1 N and lactophenol blue). The percentage of pollen viability was obtained with an optic microscope (250×) and compared with the pollen germination at culture media where one raquis of each genotype was gentle shaken in a petridish. Three replicates (petri dishes) was performed for each genotype which were maintained at the temperature of 25 °C and air humidity around 95 % for 30 min. The factors (staining methods, genotypes and times) and their interactions were evaluated by the analysis of variance, F test (P < 0.01) and the means compared by the t test (P < 0.05). The lactophenol blue staining was more sensible than the iodine staining method to detect the decrease of pollen viability which occurs naturally in sugarcane. The iodine staining method was more stable and easier than lactophenol to perform the inflorescence classification at any evaluated time (6.00, 8.00 and 9.00 a. m.). Both staining methods overestimated the viability obtained by the germination at culture media when performed at 6.00 a.m. © 2012 Society for Sugar Research & Promotion.

Effects of re-stripping on the seminal characteristics of pacu (Piaractus mesopotamicus) during the breeding season

Seminal characteristics in teleost fish with an annual reproductive period, such as pacu (Piaractus mesopotamicus), may vary during the breeding season. The sperm formed before the beginning of the spawning period may be stored for a long time, causing damage to the cells. Therefore, re-stripping may be an important way to eliminate the old and allow for the collection of new spermatozoids. In this study, we analyzed the seminal characteristics of hormonally induced pacu at the beginning, middle and end of the breeding season, and we analyzed samples from re-stripped males (stripped first at the beginning, re-stripped in the middle, and re-stripped again at the end of the season) during two breeding seasons. The sperm density, ionic composition, pH, and osmolality were similar among the groups. The semen volume, seminal plasma protein concentration and incidence of morphologically anomalous sperm increased over time. In addition, some parameters that are associated with good-quality semen decreased, such as sperm motility, viability and DNA integrity. Moreover, we observed a positive association among motility, viability and DNA integrity for sperm with elevated 11-ketotestosterone, but there was no such association for fshb or lhb mRNA levels in the pituitary. The semen that was obtained earlier (at the beginning) or from re-stripped males exhibited better characteristics than the other samples collected. In conclusion, collecting semen from pacu at the end of breeding season should be avoided; it is preferable to strip early and then re-strip later in the season, and this approach may be used for diverse aquaculture purposes.

Viability, production and morphology of pollen grains for different species in the genus Manihot (Euphorbiaceae)

(Viability, production and morphology of pollen grains for different species in the genus Manihot (Euphorbiaceae)). The objective of this work was to characterize the viability, production and morphology of pollen for different species in the genus Manihot. Floral buds from Manihot accessions were collected from two germplasm banks at Embrapa Cassava & Fruits. The viability of the pollen was assessed via colorimetric, in vitro and in vivo assays. The diameter of the pollen grains was determined by measuring the transversal length of the grain. The experimental design was entirely randomized. Studies on pollen ultrastructure were performed via scanning electron microscopy. Pollen viability was high in the colorimetric tests and intermediate in vivo tests; there was no germination in the in vitro tests. The average production for all accessions was 1,253 pollen grains per floral bud. The size of the pollen grains varied from 132 to 163 pm in the wild accessions, and 129 to 146 pm in the cultivated accessions. The pollen grains for all accessions were very large, apolar, spherical as well as inaperturate, with an exine ornamented with pila organized in a Croton pattern. The wild accessions, in general, produced more and larger pollen grains compared with the cultivated accessions.

Seed viability and dormancy mechanisms of Leucopogon melaleucoides Cunn. ex DC. (Epacridaceae)

Leucopogon melaleucoides, a flowering shrub, is desired by floricultural markets but is difficult to propagate. Seed viability was tested and dormancy mechanisms were studied to develop a commercial propagation system. Although around 56% of seed were viable, germination was completely inhibited unless the endocarp was removed. After-ripened seed (8 months after collection) germinated faster than fresh seed (2 days after collection), but germination occurred over a prolonged period (155 days). Germination of after-ripened seed was promoted with GA(3) or a commercial smoke product containing unknown plant growth regulators. All viable seed treated with GA(3) at 1000 mg L-1 had germinated after 24 days. The results suggest that both a physical and physiological dormancy mechanism occur for this species, with removal of the endocarp and pretreatment with 1000 mg L-1 GA(3) promoting complete germination of viable seed.

Fruit processing, seed viability and dormancy mechanisms of Persoonia sericea A. Cunn. ex R. Br. and P-virgata R. Br. (Proteaceae)

The morphology of the fruit and difficulties with fruit processing impose major limitations to germination of Persoonia sericea and P. virgata. The mesocarp must be removed without harming the embryo. Fermentation of fruit or manual removal of the mesocarp was effective but digestion in 32% hydrochloric acid (HCl) completely inhibited germination. The endocarp is extremely hard and therefore very difficult and time consuming to remove without damaging the seeds. The most efficient method was cracking the endocarp with pliers, followed by manual removal of seeds. Germination was completely inhibited unless at least half of the endocarp was removed. Microbial contamination of the fruit and seeds was controlled by disinfestation and germination of the seed under aseptic conditions. The results suggest that dormancy in these species is primarily due to physical restriction of the embryo by the hard endocarp.

Solid Lipid Nanoparticles For Hydrophilic Biotech Drugs: Optimization And Cell Viability Studies (caco-2 & Hepg-2 Cell Lines).

Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

Inhibitory Effects Of A Cured Antibacterial Bonding System On Viability And Metabolic Activity Of Oral Bacteria.

To evaluate the antimicrobial efficacy of Clearfil SE Protect (CP) and Clearfil SE Bond (CB) after curing and rinsed against five individual oral microorganisms as well as a mixture of bacterial culture prepared from the selected test organisms. Bacterial suspensions were prepared from single species of Streptococcus mutans, Streptococcus sobrinus, Streptococcus gordonii, Actinomyces viscosus and Lactobacillus lactis, as well as mixed bacterial suspensions from these organisms. Dentin bonding system discs (6 mm×2 mm) were prepared, cured, washed and placed on the bacterial suspension of single species or multispecies bacteria for 15, 30 and 60 min. MTT, Live/Dead bacterial viability (antibacterial effect), and XTT (metabolic activity) assays were used to test the two dentin system's antibacterial effect. All assays were done in triplicates and each experiment repeated at least three times. Data were submitted to ANOVA and Scheffe's f-test (5%). Greater than 40% bacteria killing was seen within 15 min, and the killing progressed with increasing time of incubation with CP discs. However, a longer (60 min) period of incubation was required by CP to achieve similar antimicrobial effect against mixed bacterial suspension. CB had no significant effect on the viability or metabolic activity of the test microorganisms when compared to the control bacterial culture. CP was significantly effective in reducing the viability and metabolic activity of the test organisms. The results demonstrated the antimicrobial efficacy of CP both on single and multispecies bacterial culture. CP may be beneficial in reducing bacterial infections in cavity preparations in clinical dentistry.

Genetic Breeding And Diversity Of The Genus Passiflora: Progress And Perspectives In Molecular And Genetic Studies.

Despite the ecological and economic importance of passion fruit (Passiflora spp.), molecular markers have only recently been utilized in genetic studies of this genus. In addition, both basic genetic researches related to population studies and pre-breeding programs of passion fruit remain scarce for most Passiflora species. Considering the number of Passiflora species and the increasing use of these species as a resource for ornamental, medicinal, and food purposes, the aims of this review are the following: (i) to present the current condition of the passion fruit crop; (ii) to quantify the applications and effects of using molecular markers in studies of Passiflora; (iii) to present the contributions of genetic engineering for passion fruit culture; and (iv) to discuss the progress and perspectives of this research. Thus, the present review aims to summarize and discuss the relationship between historical and current progress on the culture, breeding, and molecular genetics of passion fruit.

Microsatellite In Aeschynomene Falcata (leguminosae): Diversity, Cross-amplification, And Chromosome Localization.

Aeschynomene falcata is an important forage species; however, because of low seed production, it is underutilized as forage species. Aeschynomene is a polyphyletic genus with a challenging taxonomic position. Two subgenera have been proposed, and it is suggested that Aeschynomene can be split in 2 genera. Thus, new markers, such as microsatellite sequences, are desirable for improving breeding programs for A. falcata. Based on transferability and in situ localization, these microsatellite sequences can be applied as chromosome markers in the genus Aeschynomene and closely related genera. Here, we report the first microsatellite library developed for this genus; 11 microsatellites were characterized, with observed and expected heterozygosities ranging from 0.0000 to 0.7143 and from 0.1287 to 0.8360, respectively. Polymorphic information content varied from 0.1167 to 0.7786. The departure from Hardy-Weinberg equilibrium may have resulted from frequent autogamy, which is characteristic of A. falcata. Of the 11 microsatellites, 9 loci were cross-amplified in A. brevipes and A. paniculata and 7 in Dalbergia nigra and Machaerium vestitum. Five of these 7 cross-amplified microsatellites were applied as probes during the in situ hybridization assay and 2 showed clear signals on A. falcata chromosomes, ensuring their viability as chromosome markers.