Physiological and health consequences of social status in zebrafish (Danio rerio).

Social status affects access to food, mates and shelter and has consequences for the physiology of individuals and their health status. In the zebrafish (Danio rerio), an emerging model for studies into animal behavior, the possible consequences of social hierarchy to an individual's physiology and health are unknown. To address this, in this species we assessed the effects of social interaction (for periods of 1-5days) on growth, stress, immune function and reproductive condition. Wide-ranging differences in physiology occurred between the social ranks, some of which were sex-related and time-dependent. In both sexes, dominant fish were larger than subordinates and dominant males had a higher growth rate during the trials. Subordinates had higher plasma cortisol and in males higher telencephalic corticotrophin-releasing hormone, neuropeptide y and glucocorticoid receptor gene expression. Splenic cytokine expression suggested differences in immune status between ranks in both sexes and hematocrit was elevated in subordinate males. In both sexes, dominants and subordinates differed in the expression of genes for various gonadal sex steroid receptors and steroidogenic enzymes and in dominant females the ovary was larger relative to body mass compared with in subordinates. Dominant males had higher plasma 11-ketotestosterone than subordinates and there was an increase in the number of spermatids in their testes over the duration of the study that was not seen in subordinate males. The wide-ranging physiological differences seen between dominant and subordinate zebrafish as a consequence of their social status suggest negative health impacts for subordinates after prolonged durations in those hierarchies.

A critical analysis of the biological impacts of plasticizers on wildlife.

This review provides a critical analysis of the biological effects of the most widely used plasticizers, including dibutyl phthalate, diethylhexyl phthalate, dimethyl phthalate, butyl benzyl phthalate and bisphenol A (BPA), on wildlife, with a focus on annelids (both aquatic and terrestrial), molluscs, crustaceans, insects, fish and amphibians. Moreover, the paper provides novel data on the biological effects of some of these plasticizers in invertebrates, fish and amphibians. Phthalates and BPA have been shown to affect reproduction in all studied animal groups, to impair development in crustaceans and amphibians and to induce genetic aberrations. Molluscs, crustaceans and amphibians appear to be especially sensitive to these compounds, and biological effects are observed at environmentally relevant exposures in the low ng l(-1) to microg l(-1) range. In contrast, most effects in fish (except for disturbance in spermatogenesis) occur at higher concentrations. Most plasticizers appear to act by interfering with the functioning of various hormone systems, but some phthalates have wider pathways of disruption. Effect concentrations of plasticizers in laboratory experiments coincide with measured environmental concentrations, and thus there is a very real potential for effects of these chemicals on some wildlife populations. The most striking gaps in our current knowledge on the impacts of plasticizers on wildlife are the lack of data for long-term exposures to environmentally relevant concentrations and their ecotoxicity when part of complex mixtures. Furthermore, the hazard of plasticizers has been investigated in annelids, molluscs and arthropods only, and given the sensitivity of some invertebrates, effects assessments are warranted in other invertebrate phyla.

Atlantic salmon eggs favour sperm in competition that have similar major histocompatibility alleles.

Polyandry and post-copulatory sexual selection provide opportunities for the evolution of female differential sperm selection. Here, we examined the influence of variation in major histocompatibility (MH) class I allelic composition upon sperm competition dynamics in Atlantic salmon. We ran in vitro fertilization competitions that mimicked the gametic microenvironment, and replicated a paired-male experimental design that allowed us to compare differences in sperm competition success among males when their sperm compete for eggs from females that were genetically either similar or dissimilar at the MH class I locus. Concurrently, we measured variation in spermatozoal traits that are known to influence relative fertilization success under these conditions. Contrary to the findings demonstrating mechanisms that promote MH complex heterozygosity, our results showed that males won significantly greater relative fertilization success when competing for eggs from genetically similar females at the MH class I. This result also showed covariation with the known influences of sperm velocity on relative fertilization success. We discuss these unexpected findings in relation to sperm-egg recognition and hybridization avoidance mechanisms based upon immunogenetic variation.

Variability in measures of reproductive success in laboratory-kept colonies of zebrafish and implications for studies addressing population-level effects of environmental chemicals.

Laboratory tests that quantify reproductive success using model fish species are used to investigate for population-level effects of endocrine disrupting chemicals (EDCs) and other chemicals discharged into the environment. Even for the zebrafish (Danio rerio), however, one of the most widely used laboratory models, surprisingly little is known about the normal variability in measures of reproductive success and this information is crucial for robust test design. In this study, the dynamics of breeding and inherent variability in egg output/viability and sperm quality were characterized among individuals/colonies and over time in 34 colonies of laboratory-kept zebrafish over a 20-day study period. For this work, a '6 x 6' (six males and six females) colony size was adopted, as this is both environmentally relevant and optimal when considering egg output and animal welfare combined: an initial experiment showed egg output per female increased with decreasing colony size however, there was also a parallel increase in aggressive behavior. Both egg output and viability in '6 x 6' colonies were highly variable among colonies (with co-efficients of variation (CVs) of 30 and 11%, respectively) and over the 20-day study duration (considering egg output and viability of all the colonies combined, the CVs were 20 and 12%, respectively). The patterns of egg production also differed among the '6 x 6' colonies, and they included a cyclical output, a consistent daily output, an infrequent egg output with intermittent days of very high egg output, and an output with no obvious pattern. Sperm quality, measured as percentage motility and curvilinear velocity (VCL), was variable both among individuals within '6 x 6' colonies and across colonies, with percentage motility being the most variable parameter (mean CVs of 82% inter-individual within colonies and 49% inter-colony). Sperm quality did not, however, vary over a 24h period. A minimum number of six replicate '6 x 6' colonies, assessed daily for a period of 4 days, was required per treatment to detect a 40% change in egg output. The minimum numbers of individual males required per treatment to detect a 40% change in sperm quality using the breeding system adopted were 32 males for percentage motility and 12 males for VCL, equivalent to six and two '6 x 6' colonies, respectively. These data demonstrate the need for high levels of replication when testing for effects of EDCs on reproductive output in the zebrafish model in an environmentally relevant ('6 x 6') breeding matrix.

Gonadal transcriptome responses and physiological consequences of exposure to oestrogen in breeding zebrafish (Danio rerio).

Environmental oestrogens are widespread in the aquatic environment and cause alterations in sexual development and function in vertebrates. The molecular pathways underpinning these effects, however, remain poorly understood. In this study, we aimed at generating a mechanistic understanding of the disruptive effects of exposure to environmentally relevant concentrations of 17 alpha-ethinyloestradiol (EE(2)) on reproduction in zebrafish, by anchoring the transcriptomic alterations induced with the physiological consequences of exposure. Breeding colonies of zebrafish were exposed for a 21-day period to three concentrations of EE(2) (0.05, 0.5 and 5 ng/L) and the gonadal transcriptomic alterations induced (determined using a 17,000 oligonucleotide microarray) were analysed together with physiological effects seen on reproductive output of both males and females. Exposure to 5 ng EE(2)/L resulted in reproductive impairment characterised by a decrease in egg production, alterations in sperm quality and reduced fertilisation success. The effects seen were associated with altered expression of 114 and 131 genes in the gonads of males and females, respectively. The biological processes most affected by the exposure were protein metabolism in males and mitochondria organisation and biogenesis in females. Genes involved in the regulation of cell cycle progression, the ubiquitin system and glutathione peroxidase were affected by the EE(2) exposure and associated with the changes observed in gamete quality in both genders. In summary, we demonstrated that EE(2) exposure compromised the reproductive health of breeding zebrafish at environmentally relevant concentrations. The molecular mechanisms mediating some of these effects were identified and included those impacting processes central to gametogenesis in both males and females.

Dimorphic sperm and the unlikely route to fertilisation in the yellow seahorse.

Uniquely among vertebrates, seahorses and pipefishes (Family Syngnathidae) incubate their eggs within a male brood pouch. This has contributed to a widespread, but poorly founded belief, that the eggs are fertilised using spermatozoa that are deposited directly into the brood pouch via an internal sperm duct. Anatomical dissections showed, however, not only that direct sperm deposition into the pouch is physically impossible, but that spermatozoa must somehow travel a significant distance (>4 mm) outside the body of the male, to reach and fertilise eggs in the pouch. Observations of courtship and mating behaviour also revealed that the pouch closes immediately after mating, and that sperm transfer must occur within a time window of no more than 6 s. In addition to this, the yellow seahorse produces extraordinarily low quantities of dimorphic spermatozoa, but is nevertheless highly fertile and can produce broods that exceed 100 embryos. The entire fertilisation process in seahorses is therefore uniquely efficient among vertebrates, yet paradoxically involves several steps that would seem to complicate, and even appear to prevent, the interaction of the gametes. Although we are still unable to describe the exact fertilisation mechanism, we speculate that spermatozoa are ejaculated into a mixture of ovarian fluid and eggs, while the male and female are in close contact. Thereafter, this mixture must enter the pouch, whereupon the spermatozoa encounter seawater. These observations also support the view, indirectly inferred in previous publications, that sperm competition in seahorses is not only non-existent but impossible.

Stickleback sperm saved by salt in ovarian fluid.

Sperm of the three-spined stickleback Gasterosteus aculeatus display a prolonged motility in the presence of ovarian fluid. The ovarian fluid prolongs sperm motility in freshwater from approximately 1 min to several hours, a trait that possibly gives the stickleback its unusual ability to spawn in waters of all salinities. The aim of the study was to look for factor(s) within the ovarian fluid responsible for prolonging sperm motility as well as to investigate the possible biological importance of the ovarian fluid under natural conditions. To that end, we measured the ionic content (Na(+), Cl(-), Ca(2+) and K(+)) of the ovarian fluid and prepared ionic artificial ovarian fluids. We also prepared a mannitol solution with the same osmolality as the ovarian fluid in order to distinguish between the ionic and osmotic effect. We found that the ionic artificial fluids were equally effective as the natural ovarian fluid in prolonging sperm motility and survival over a range of concentrations, whereas the mannitol solution was far less effective. By insertion of natural ovarian fluid or ovarian fluid from which macromolecules had been removed by ultra filtration in nests it was found that macromolecules help by retaining ions. We also found that ovarian fluid in water, at concentrations as low as 0.75 and 1.56%, prolonged sperm motility and that the concentration of ions (Na(+)) present in the nest 15 min after spawning corresponded to at least 3% ovarian fluid. Previous fertilisation experiments have shown that it takes at least 15 min for stickleback sperm to fertilise all eggs in a batch. This indicates that the role of ovarian fluid in prolonging the sperm motility is biologically relevant and that the effect is exerted by the fluid's ionic content.

Effect of parental age and associated size on fecundity, growth and survival in the yellow seahorse Hippocampus kuda.

Seahorses, together with the pipefishes (Family Syngnathidae), are the only vertebrates in which embryonic development takes place within a specialised body compartment, the brood pouch, of the male instead of the female. Embryos develop in close association with the brood pouch epithelium in a manner that bears some resemblance to embryo-placental relationships in mammals. We have explored the hypothesis that parental body size and age should affect offspring postnatal growth and survival if brood pouch quality impacts upon prenatal embryonic nutrition or respiration. Using an aquarium population of the yellow seahorse, Hippocampus kuda, we show here that large parents produce offspring whose initial postnatal growth rates (weeks one to three) were significantly higher than those of the offspring of younger and smaller parents. Whereas 90% of offspring from the larger parents survived for the duration of the study (7 weeks), less that 50% of offspring from smaller parents survived for the same period. For the offspring of large parents, growth rates from individual males were negatively correlated with the number of offspring in the cohort (r=-0.82; P<0.05); this was not the case for offspring from small parents (r=0.048; P>0.9). Observations of embryos within the pouch suggested that when relatively few embryos are present they may attach to functionally advantageous sites and thus gain physiological support during gestation. These results suggest that male body size, and pouch size and function, may influence the future fitness and survival of their offspring.

Concepts in sperm heterogeneity, sperm selection and sperm competition as biological foundations for laboratory tests of semen quality.

Stringent selection mechanisms, in both internal and external fertilisation systems, reject all but a significant minority of the spermatozoa released at ejaculation. Sperm competition theory provides circumstantial evidence that the selection process involves mechanisms by which the quality of the fertilising spermatozoon is controlled, thereby ensuring that females and their offspring receive high quality genetic material. In this review we examine some of these selection processes to see whether they could be exploited for the improvement of laboratory tests of sperm quality. Such tests are not only required for clinical and agricultural purposes, but are increasingly needed in fields such as reproductive and environmental toxicology where the species requirement is much broader. Despite many years of research, sperm quality assessment methods continue to provide imprecise data about fertility; here we suggest that this may be a consequence of using tests that focus on the spermatozoa that would normally be unable to fertilise under natural conditions. To achieve fertilisation a spermatozoon must be capable of responding appropriately to external signalling stimuli; those involving protein kinase-regulated flagellar function seem especially influential in governing effects ranging from non-Mendelian inheritance in mammals to sperm chemotaxis in sea urchins. Examination of the elicited responses reveals considerable heterogeneity in all species. Here we propose that this level of heterogeneity is meaningful both in terms of understanding how spermatozoa from some individuals possess fertility advantages over spermatozoa from their rivals in sperm competition, and in that the heterogeneity should be exploitable in the development of more accurate laboratory tests.