Inbreeding and genetic structure in the endangered Sorraia horse breed: implications for its conservation and management

The Sorraia horse is a closed breed with reduced effective population size and considered in critical maintained risk status. The breed exists in 2 main breeding populations, one in Portugal and one in Germany, with a smaller population size. A set of 22 microsatellite loci was used to examine genetic diversity and structure of the Sorraia horse breed and to compare individual inbreeding coefficient F, estimated from pedigree data, with individual heterozygosity and mean d(2). The Sorraia horse shows lower levels of microsatellite diversity when compared with other horse breeds. Due to management strategies, there are clear differences in the genetic structure of the 2 main Sorraia horse populations. Individual heterozygosity was shown to be a good estimator, used together with or as an alternative to inbreeding coefficient, in predicting fitness and evaluating the inbreeding level of the Sorraia horse. The information gathered in this study, combined with information available from previous studies, offers an important and wide information base for the future development of an effective breeding management of the Sorraia horse in order to preserve this endangered breed.     

SNARE proteins and caveolin-1 in stallion spermatozoa: possible implications for fertility

Proteins implicated in the "SNARE hypothesis" for membrane fusion have been characterized in the acrosome of several mammalian species, and a functional role for these proteins during the acrosome reaction has been proposed. We have investigated the presence of SNAREs in equine sperm, using semen samples obtained from stallions with varying fertility. Immunocytochemical analysis revealed that members of different SNARE families can be detected on the acrosome of equine sperm, notably in the acrosomal cap and equatorial segment. These proteins include the t-SNARE syntaxin, the v-SNARE synaptobrevin/VAMP, the calcium sensor synaptotagmin, and the ATPase NSF. Also present is caveolin-1, a component of lipid rafts. Stallions with fertility problems presented the worst quality of sperm and acrosomal membrane, and had less sperm cells stained positively for SNAREs and caveolin-1, than sperm from fertile donors (p < 0.001). Ubiquitin surface staining was also performed and it seemed to inversely correlate with stallion fertility, supporting data obtained with the negative staining technique. A male-related problem was confirmed when mares that had failed to impregnate with samples from an infertile stallion were successfully inseminated with sperm from a fertile donor. Furthermore NSF, synaptotagmin and caveolin-1 staining seemed to be useful in predicting stallion fertility, i.e. significantly more sperm cells stained positively for these proteins in samples from fertile males. Although these results need to be expanded on a larger scale, they suggest that acrosomal and surface staining of equine sperm with novel probes may constitute useful tools in predicting stallion fertility.     

Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse

In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca2+]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca2+]i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca2+]i oscillations to injection of stallion sperm, and [Ca2+]i monitoring was performed. Such pairs consistently displayed [Ca2+]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species.     

Genetic parameters for production,health, fertility and longevity traits in dairy cows

Milk production, fertility, longevity and health records, were extracted from databases of two milk recording organisations in the United Kingdom for the first three lactations of the Holstein–Friesian breed. These included data related to health events (mastitis and lameness), voluntarily recorded on a proportion of farms. The data were analysed to calculate disease incidence levels and to estimate genetic parameters for health traits and their relationships with production and other functional traits. The resulting dataset consisted of 124 793 lactations from 75 137 animals of 1586 sires, recorded in 2434 herds. Incidence of health events increased with parity. The overall incidence of mastitis (MAS) and lameness (LAM), defined as binary traits, were 17% and 16%, respectively. Heritability estimates for MAS and LAM were 0.04 and 0.02, respectively, obtained from repeatability linear sire models. Heritability estimates of mastitis and lameness as count traits were slightly higher, 0.05 and 0.03, respectively. Genetic correlations were obtained by bivariate analyses of all pair-wise combinations between milk 305-day yield (MY), protein 305-day yield (PY), fat 305-day yield (FY), lactation average log_e transformed lactation average somatic cell count (SCS), calving interval (CI), days to first service (DFS), non-return at 56 days (NR56), number of inseminations (NINS), mastitis (MAS), number of mastitis episodes (NMAS), lameness (LAM), number of lameness episodes (NLAM) and lifespan score (LS). As expected, MAS was correlated most strongly with SCS (0.69), which supports the use of SCS as an indicator trait for mastitis. Genetic correlations between MAS and yield and fertility traits were of similar magnitude ranging from 0.27 to 0.33. Genetic correlations between MAS with LAM and LS were 0.38 and −0.59, respectively. Not all genetic correlations between LAM and other traits were significant because of fewer numbers of lameness records. LAM had significant genetic correlations with MY (0.38), PY (0.28), CI (0.35), NINS (0.38) and LS (−0.53). The heritability estimates of mastitis and lameness were low; therefore, genetic gain through direct selection alone would be slow, yet still positive and cumulative. Direct selection against mastitis and lameness as additional traits should reduce incidence of both diseases, and simultaneously improve fertility and longevity. However, both health traits had antagonistic relationships with production traits, thus genetic gain in production would be slower.     

Oxidative stress, osmotic stress and apoptosis: impacts on sperm function and preservation in the horse

Oxidative stress is an important component of the cytopathology of equine spermatozoa undergoing storage as liquid or frozen semen. Damage to chromatin, membranes and proteins of sperm are important components of oxidative damage to sperm. Similarly, sperm are exposed to a variety of osmotic stresses during storage that result from exposure to hypertonic media or result as a consequence of osmotic changes induced during freezing. A number of changes induced during processing and storage of equine sperm also appear to induce apoptotic-like changes which may adversely affect sperm survival and function. These processes appear in many cases to be interrelated, and this review will examine current understanding of these processes on the equine sperm function.     

The quantification of lipid and protein oxidation in stallion spermatozoa and seminal plasma: seasonal distinctions and correlations with DNA strand breaks, classical seminal parameters and stallion fertility

The goal of this work was to correlate oxidative stress caused by reactive oxygen species (ROS) and DNA damage with classic semen parameters in spermatozoa and seminal plasma of fertile and subfertile stallions. Oxidation was measured in both lipids and proteins, using the thiobarbituric acid reactive species (TBARS) assay and the DNPH carbonyl groups assay, respectively. Sperm DNA damage was monitored using the TUNEL assay. These parameters were monitored in samples obtained during the breeding and the non-breeding seasons. In general, fertile stallions showed better classical semen parameters, and those parameters improved from the non-breeding to the breeding season, although an increase in sperm production was accompanied by a decrease in the semen quality from subfertile stallions in the breeding season. In terms of oxidation levels we found that there were clear differences whether lipids or proteins were considered. In the breeding season there seemed to be a tendency towards normalizing lipid oxidation in spermatozoa and seminal plasma, and protein oxidation in the seminal plasma, of both fertile and subfertile animals. Thus, differences monitored in the non-breeding season were no longer visible. Interestingly, a higher level of protein oxidation was found in the sperm of fertile animals in the breeding season. Considering that there were positive correlations between sperm protein oxidation and sperm motility and vitality, these results suggests that the oxidation of semen proteins may be important for sperm function. On the other hand, lipid oxidation in the seminal plasma seemed to be a general indicator for sperm damage. In the non-breeding season positive correlations between lipid and protein oxidation levels in both sperm and seminal plasma and several defects in sperm function were found, but only for subfertile animals, thus suggesting that lipid and protein oxidation may aid in the identification of subfertile stallions during the non-breeding season. Levels of ROS production never seemed to result in compromised sperm DNA integrity, indicating that measurements were within physiological levels and/or that there is an efficient antioxidant activity in stallion sperm cells.     

Channeling in native microbial pathways: Implications and challenges for metabolic engineering

Intracellular enzymes can be organized into a variety of assemblies, shuttling intermediates from one active site to the next. Eukaryotic compartmentalization within mitochondria and peroxisomes and substrate tunneling within multi-enzyme complexes have been well recognized. Intriguingly, the central pathways in prokaryotes may also form extensive channels, including the heavily branched glycolysis pathway. In vivo channeling through cascade enzymes is difficult to directly measure, but can be inferred from in vitro tests, reaction thermodynamics, transport/reaction modeling, analysis of molecular diffusion and protein interactions, or steady state/dynamic isotopic labeling. Channeling presents challenges but also opportunities for metabolic engineering applications. It rigidifies fluxes in native pathways by trapping or excluding metabolites for bioconversions, causing substrate catabolite repressions or inferior efficiencies in engineered pathways. Channeling is an overlooked regulatory mechanism used to control flux responses under environmental/genetic perturbations. The heterogeneous distribution of intracellular enzymes also confounds kinetic modeling and multiple-omics analyses. Understanding the scope and mechanisms of channeling in central pathways may improve our interpretation of robust fluxomic topology throughout metabolic networks and lead to better design and engineering of heterologous pathways.     

Assembling the fungal tree of life: progress, classification, and evolution of subcellular traits

Based on an overview of progress in molecular systematics of the true fungi (Fungi/Eumycota) since 1990, little overlap was found among single-locus data matrices, which explains why no large-scale multilocus phylogenetic analysis had been undertaken to reveal deep relationships among fungi. As part of the project "Assembling the Fungal Tree of Life" (AFTOL), results of four Bayesian analyses are reported with complementary bootstrap assessment of phylogenetic confidence based on (1) a combined two-locus data set (nucSSU and nucLSU rDNA) with 558 species representing all traditionally recognized fungal phyla (Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota) and the Glomeromycota, (2) a combined three-locus data set (nucSSU, nucLSU, and mitSSU rDNA) with 236 species, (3) a combined three-locus data set (nucSSU, nucLSU rDNA, and RPB2) with 157 species, and (4) a combined four-locus data set (nucSSU, nucLSU, mitSSU rDNA, and RPB2) with 103 species. Because of the lack of complementarity among single-locus data sets, the last three analyses included only members of the Ascomycota and Basidiomycota. The four-locus analysis resolved multiple deep relationships within the Ascomycota and Basidiomycota that were not revealed previously or that received only weak support in previous studies. The impact of this newly discovered phylogenetic structure on supraordinal classifications is discussed. Based on these results and reanalysis of subcellular data, current knowledge of the evolution of septal features of fungal hyphae is synthesized, and a preliminary reassessment of ascomal evolution is presented. Based on previously unpublished data and sequences from GenBank, this study provides a phylogenetic synthesis for the Fungi and a framework for future phylogenetic studies on fungi.     

Application of assisted reproduction for population management in felids: the potential and reality for conservation of small cats

Assisted reproductive technology (ART), using the primary applied tools of AI, ET, and sperm and embryo cryopreservation, has been promoted over the past decades for its potential to conserve endangered wildlife, including felids. However, if the goal is efficient, consistent production of viable offspring for population management, then the 'potential' of ART has yet to become 'reality' for any non-domestic cat species. For the five small-sized felids (i.e., Brazilian ocelot, fishing cat, Pallas' cat, Arabian sand cat, black-footed cat) managed by Species Survival Plans (SSPs) in North American zoos, achieving this potential may be an absolute necessity if genetically viable captive populations are to be maintained into the next century. Modeling programs suggest that current SSP populations are not sustainable without periodic introduction of new founders and improved demographic parameters, including longer generation intervals and larger population sizes. ART provides the means to address each of these management challenges. In each small cat SSP species, fecal hormone metabolite assays and seminal analysis have proven useful for characterizing basal reproductive parameters, a necessary prerequisite to developing ART. Of the five SSP species, ART has been used to produce living offspring only in the ocelot, including after AI with frozen-thawed spermatozoa and following transfer of frozen-thawed IVF embryos. The true efficacy of these techniques, however, is still unknown. To improve the applicability of ART for population management, priorities for immediate research include further investigation of ovarian stimulation protocols, sperm and embryo cryopreservation methods, embryo culture systems, and fetal and neonatal viability following ART.     

Radiographic contrast medium for uterine insemination in the bitch, and its effect upon the quality and fertility of fresh dog semen

Four ejaculates were collected from each of 6 adult male beagle dogs. The second fraction was divided into 3 aliquants which were then diluted with physiological saline, an isoosmolar solution of sodium/meglumine diatroate, and an iso-osmolar solution of iohexol. The diluted samples were incubated at 39 degrees C and evaluated at 0, 60, 90, 120, 240 and 360 min after dilution. A variety of assessments was made, including, spermatozoal motility, spermatozoal morphology, and acrosorne status. The practicality of using 1.0 ml of iso-osmolar contrast medium combined with radiographic examination was evaluated as a method of confirming accurate placement of a transcervical uterine catheter by injecting contrast after positioning the catheter in 4 beagle bitches. The effect of the procedure on fertility was assessed using 5 greyhound bitches which were inseminated with fresh semen and in which pregnancy was monitored using diagnostic B-mode ultrasound imaging. There was no significant difference between physiological saline and the sodium/meglumine diatroate solution upon semen quality, while the iohexol solution produced a significant reduction in spermatozoal motility and morphology. No adverse clinical effects were observed when contrast medium was administered into the uterus to either group of bitches. A subjective assessment of radiographic quality showed that the sodium/meglumine diatroate solution, which contained twice the iodine concentration of the iohexol solution, produced significantly greater radiopacity and was radiographically more useful than the iohexol solution. The sodium/meglurnine diatroate solution had no adverse effect upon the fertility of dog semen, and all bitches that were inseminated with this technique conceived and maintained the pregnancy to term. Litter size was considered to be normal for the breed. Small volumes of an iso-osmolar solution of sodium/meglumine diatroate may be useful for ensuring correct placement of transcervical catheters prior to artificial insemination in the bitch.     

Selection for litter size and litter birthweight in Large White pigs: Maximum,mean and variability of reproduction traits

Gradually increasing trend of litter size poses a challenge to pig farmers in terms of managing larger litters. Therefore, it seems that a balanced approach that optimises litter size, litter birthweight, and uniformity of those traits is needed in order to address animal welfare and farm management concerns. This study aimed to investigate this issue by defining several traits for total number born (TNB), number born alive (NBA) and litter birthweight (LW). First, the highest value from at least five records per sow was selected as maximum (max) value for each reproduction trait. Second, a mean (mean) for each reproduction trait was calculated per sow. Last, the variability of reproduction traits between parities of the sow was calculated as log-transformed variance of residuals of all observations per sow for each reproduction trait (LnVar). In total, 23 193 Large White sows from Topigs Norsvin with 152 282 litter records were used for analysis in ASReml 4.1. Also, a simulation of breeding schemes was performed with the use of SelAction 2.1 and estimates from genetic analysis. Maximum value of reproductive traits had a much higher heritability than repeated observations or mean of reproduction traits, e.g., 0.31 for maxTNB vs. 0.12 for TNB and 0.07 for meanTNB, which allows for a faster response under selection. The maximum value traits, however, were found to carry more risks, i.e. higher ratio of stillborn (not for maxNBA) and increased variability of traits. Thus, using them in breeding programme should be carefully considered. The genetic coefficient of variation on SD level estimated to indicate the genetic magnitude for variability phenotypes indicated a maximum change of 6–9% in genetic SD of TNB, NBA and LW. The genetic correlations between mean and corresponding variability traits varied from 0.66 to 0.74, whereas the correlation between other mean and variability traits ranged from 0.33 to 0.99. The simulation indicated that even with selection targeted against the variability of reproduction traits, a very limited change should be expected due to a complex genetic and phenotypic relationship between the traits. In the scenarios with selection against LnVarTNB and LnVarLW, this was a decrease of 0.1–0.6% per year, whereas in scenario with selection against LnVarNBA, the range was 0.6–1.1% per year. It is still possible to increase litter size and birthweight further, however, a balance between mean and variability of reproduction traits is required, which can be obtained only by a very well designed breeding programme.     

Correlated responses to selection for faster development and early reproduction in Drosophila: the evolution of larval traits

Studies on selection for faster development in Drosophila have typically focused on the trade-offs among development time, adult weight, and adult life span. Relatively less attention has been paid to the evolution of preadult life stages and behaviors in response to such selection. We have earlier reported that four laboratory populations of D. melanogaster selected for faster development and early reproduction, relative to control populations, showed considerably reduced preadult development time and survivorship, dry weight at eclosion, and larval growth rates. Here we study the larval phase of these populations in greater detail. We show here that the reduction in development time after about 50 generations of selection is due to reduced duration of the first and third larval instars and the pupal stage, whereas the duration of the second larval instar has not changed. About 90% of the preadult mortality in the selected populations is due to larval mortality. The third instar larvae, pupae, and freshly eclosed adults of the selected populations weigh significantly less than controls, and this difference appears during the third larval instar. Thereafter, percentage weight loss during the pupal stage does not differ between selected and control populations. The minimum amount of time a larva must feed to subsequently complete development is lower in the selected populations, which also exhibit a syndrome of reduced energy expenditure through reduction in larval feeding rate, larval digging and foraging activity, and pupation height. Comparison of these results with those observed earlier in populations selected for adaptation to larval crowding and faster development under a different protocol from ours reveal differences in the evolved traits that suggest that the responses to selection for faster development are greatly affected by the larval density at which selection acts and on details of the selection pressures acting on the timing of reproduction.     

The effect of selection for fertility of frozen-thawed semen on spermatozoa oxygen uptake, motility and concentration, and ejaculate volume in the chicken

The response to 3 generations of selection for duration of fertility of frozen-thawed semen on fertility and hatchability of fresh and frozen-thawed semen, spermatozoa oxygen uptake, motility and concentration, and ejaculate volume was measured in a broiler line of chickens. The selected line, as compared to the control, had significantly higher levels for all fertility traits of frozen-thawed semen but not hatchabilities. For fresh semen, the lines differed only for duration of fertility. The only difference in semen quality traits was in oxygen uptake by spermatozoa of frozen-thawed semen. The differences between lines for the other parameters were not significant but were in a direction that supported the hypothesis that selection had resulted in improved reproductive capacity. All estimates of fertility within an ejaculate were positively correlated (P < 0.01). Correlations between corresponding fertility estimates of fresh and frozen-thawed semen were positive, low and generally non-significant. The proportion of variation as determined by stepwise regression, in fertility estimates accounted for by the measurements of oxygen uptake and motility of fresh and frozen-thawed spermatozoa, ejaculate volume and spermatozoa concentration ranged from 12 to 19 percent.     

Framing the ethical and legal issues of human artificial gametes in research,therapy, and assisted reproduction: A German perspective

Recent results from studies on animals suggest that functional germ cells may be generated from human pluripotent stem cells, giving rise to three possibilities: research with these so‐called artificial gametes, including fertilization experiments in vitro; their use in vivo for therapy for the treatment of human infertility; and their use in assisted reproductive technologies in vitro. While the legal, philosophical, and ethical questions associated with these possibilities have been already discussed intensively in other countries, the debate in Germany is still at its beginning. A systematic and detailed analysis of the legal framework in Germany is provided with regard to the three possibilities, including the applicable statutory laws as well as the constitutional law. The question emerges as to whether the statutory laws as well as the constitution justify a distinction to be made between embryos of artificial and natural origin. This question is subject to philosophical analysis, discussing the distinction between person and thing, dignity and price, personality and property, and nature and technique. As a result, the criterion of naturalness alone may not be sufficient to differentiate between embryos of natural and artificial origin, and other criteria need to be identified.     

The integrative value of conflict and dispute: Implications for defining community in the native title context

Claimants and industry professionals frequently view conflict that arises in the course of a native title claim as a detriment to timely claims resolution. I argue instead that disputation itself may constitute an integrative social process through which participants define, delimit and reproduce community. I show also how ethnographic analysis of disputation can provide useful insights into broader social and cultural practices and normative value systems. Drawing on theoretical and methodological concerns with human agency and the integrative and constitutive role of conflict that challenges consensus models of community, I develop a practice‐centred approach that is attentive to the ways that participants in a dispute articulate and contest the definition of community. The possibility of viewing 'culture' and group cohesion as contingent and emergent through disputation should assist anthropologists working in the native title field to incorporate conflict as a productive aspect of social and cultural practice.     

Ant interactions with native and exotic seeds in the Patagonian steppe: Influence of seed traits,disturbance levels and ant assemblage

Invasive plants may establish strong interactions with species in their new range which could limit or enhance their establishment and spread. These interactions depend upon traits of the invader and the recipient community, and may alter interactions among native species. In the Patagonian steppe we studied interactions of native ant assemblages with seeds of native and exotic plants, and asked whether ant–seed interactions differ with seed types and disturbance levels and whether the amount and type of ant–seed interactions can be predicted if both ant and seed traits are known. To characterize and quantify ant–seed interactions, we offered baits with large seeds of Pappostipa speciosa (native) and medium-sized elaiosome-bearing seeds of Carduus thoermeri (exotic), near and far from a road (high vs. low disturbed areas), and compared ant abundance and composition between areas. Interaction frequency was the highest for C. thoermeri seeds far from the road. Composition of ants interacting with C. thoermeri in these areas differed from that near the road and from that interacting with native seeds. Ant composition and abundance were similar between areas, but some species interacted more with exotic seeds in low disturbed areas. Ant foraging type predicted ant–seed interactions since the abundance of seed harvesters was positively correlated to interactions with P. speciosa, and that of generalists and predators, with interactions with C. thoermeri. The high interaction of ants with exotic seeds in low invaded areas suggests that ant activity could influence plant invasion, either by predating or dispersing seeds of invasive plants.     

Not novel, just better: competition between native and non-native plants in California grasslands that share species traits

Invasive plants have often been shown to possess novel traits such as the ability to fix nitrogen, access unused resource pools, or the ability to exude allelopathic chemicals. We describe a case of a successful invasion where the native and non-native species are very similar in most life-history characteristics including their growth forms, lifespan, and degree of summertime activity. Data from permanent transects suggest that exotic perennial grass invaders can establish into intact native-dominated grasslands, achieving cover values from 6 to 71% over several years. We also established a 4-year competition experiment to test the effect of each group—the native and non-native perennial grasses—on the other. Competitive interactions were found to consistently favor the non-native grasses: native perennial grass productivity was significantly lower in plots with exotic perennial grasses as compared to plots without exotic perennial grasses. By contrast, productivity of the exotic perennial grasses was not reduced by the presence of the native perennial grasses. These results suggest that competitive ability, rather than a unique trait, has contributed to the success of the exotic perennial grasses in our system. Management tools to control exotic perennial grass invasions are likely to negatively influence native perennial grass populations, as strategies that succeed against the invasive species may kill or reduce the native species as well.