Implementing artificial insemination as an effective tool for ex situ conservation of endangered avian species

Approximately 503 of the known species of birds are classified as ‘endangered’ or ‘critical’. Captive propagation programs have proven useful in maintaining genetic diversity and restoring wild populations of certain species, including the Peregrine falcon, California condor and Whooping crane. Artificial insemination (AI) has the potential of solving problems inherent to reproductive management of small, closed populations of endangered birds, including dealing with demographic instability, physical and behavioral disabilities, sexual incompatibility, lack of synchrony, and need to maintain gene diversity. In this review, we address the necessary methods and factors that allow AI to be applied effectively to manage rare bird populations. It is clear that semen availability and quality are the greatest limiting factors to implementing consistently successful AI for birds. Behavioral sensitivity to animal handling and the ability to minimize stress in individual birds also are keys to success. Multiple, deep vaginal inseminations can improve fertility, particularly when semen quality is marginal. Laparoscopic methods of semen transfer also have produced fertile eggs. All of these practices leading to successful AI remain dependent on having adequate basic knowledge on female reproductive status, copulatory behavior, endocrine profiles and duration of fertility, especially as related to oviposition. The overall greatest challenge and highest priority is defining these normative traits, which are highly species-specific.     

The species survival plan and the conference on reproductive strategies for endangered wildlife

The application of advanced reproduction technology to the breeding of vanishing species in captivity, as planned by the Species Survival Plan (SSP) of the American Association of Zoological Parks and Aquariums, may be crucial to practical longterm propagation of some species. However, current techniques are usually specific to domestic animals and man. Major modifications and new approaches, on a species-specific basis, will be required for broad application to specialized wild animals. Prerequisites include basic studies of unique species' physiology and basic care requirements. The current SSP program needs a major research component.     

Construction of a California condor BAC library and first-generation chicken-condor comparative physical map as an endangered species conservation genomics resource

To support genomic analysis of the endangered California condor (Gymnogyps californianus), a BAC library (CHORI-262) was generated using DNA from the blood of a female. The library consists of 89,665 recombinant BAC clones providing approximately 14-fold coverage of the presumed approximately 1.48-Gb genome. Taking advantage of recent progress in chicken genomics, we developed a first-generation comparative chicken-condor physical map using an overgo hybridization approach. The overgos were derived from chicken (164 probes) and New World vulture (8 probes) sequences. Screening a 2.8x subset of the total library resulted in 236 BAC-gene assignments with 2.5 positive BAC clones per successful probe. A preliminary comparative chicken-condor BAC-based map included 93 genes. Comparison of selected condor BAC sequences with orthologous chicken sequences suggested a high degree of conserved synteny between the two avian genomes. This work will aid in identification and characterization of candidate loci for the chondrodystrophy mutation to advance genetic management of this disease.     

The distribution of Iberian gypsophilous flora as a criterion for conservation policy

Within the EU territory, gypsum habitats with a peculiar flora are exclusively restricted to the eastern half of the Iberian Peninsula. The Habitats Directive considers the scrublands belonging to the Gypsophiletalia order as priority habitats (habitats 1,520). Although these scrublands do not represent the only kind of vegetation associated with gypsum outcrops, they tend to occur together with other types of communities that grow exclusively on this substrate. As far as vascular flora is concerned, there are some species that grow exclusively on Iberian gypsums and are accordingly included in the Spanish Red List. Besides, given the fragmentary character and punctuated location of these outcrops, the protection of this Iberian habitat and its flora is, therefore, an enormous challenge for any preservation policy. The disjunct distribution of the flora faithfully reflects this geological feature of gypsum areas. Consequently, a proper and detailed knowledge of the distribution of the gypsophilous vascular flora can be very useful in formulating an efficient preservation policy for these habitats. Using the checklist of the Iberian gypsophilous flora, we collected a wide data base with records of distribution arranged into 10 × 10 km UTM grids. For each of the 1,241 grids where at least one gypsophyte was recorded we reckoned a series of parameters in order to determine the priority level of the flora for preservation purposes. These parameters were specific richness, gypsophily level, continuous and discontinuous rarity and endangered level. Our analyses reveal serious gaps in the Spanish network of protected sites with gypsophilous flora, the most important being located in the Hoya de Baza.     

Behavior as a tool for welfare improvement and conservation management in the endangered lizard (Gallotia bravoana)

The study assessed the behavior of Gallotia bravoana (La Gomera, Canary Islands, one of the world's most threatened reptiles) to facilitate management decisions and improve welfare during initial stages of a species' recovery plan. The study analyzed—and remote-controlled video cameras filmed—the behavior of lizards housed in 3 outdoor enclosures, from May to September 2000. Daily activity showed a bimodal or unimodal pattern, all specimens basking more during June, with the largest male was the most active. Adults ate a higher percentage of vegetable items; subadults, a comparatively larger proportion of larvae and adult insects. The study analyzed individual compatibility; all interacted during 2 short periods. Aggression occurred mainly between males; because most directed high-intensity aggression toward a specific lizard, the study recommended isolating this lizard in a separate enclosure. The study recommended keeping 2 male-female pairs, who demonstrated social tolerance, as breeding pairs in separated terraria. Typical courtship behavior (head-bob sequences) occurred mainly during July, with highest frequency by the oldest male. Behavioral assessment provided data for improving the individuals' welfare in the enclosures and to select specific pairs for breeding.     

A pragmatic and utilitarian view of species translocation as a tool in conservation biology

We discuss the controversial issue of species translocation for conservation, i.e. the intentional moving of a species to a non-native area when the species is threatened by extinction in its native area. Based on an argument inspired by utilitarianism: “a conservation method is good when the number of global extinctions it prevents exceeds the number of global extinctions it causes”, we identify and briefly discuss the answer to two critical research questions, and conclude that translocation has the potential to reduce net decline in global species diversity and therefore should be accepted more widely as a tool for conservation.     

Genetic fingerprinting of germplasm accessions as an aid for species conservation: a case study with Borderea chouardii (Dioscoreaceae), one of the most critically endangered Iberian plants

BACKGROUND AND AIMS: Molecular markers have changed previous expectations about germplasm collections of endangered plants, as new perspectives aim at holding a significant representation of all the genetic diversity in the studied species to accomplish further conservation initiatives successfully. Borderea chouardii is a critically endangered allotetraploid dioecious member of Dioscoreaceae, known from a single population in the Iberian pre-Pyrenees. This population was reported to be highly structured into two genetically distinct groups of individuals corresponding to their spatial separation along the vertical cliff where it grows. In 1999, the Spanish Government of Aragón launched the first conservation programme for the ex situ preservation of this species, and since then a seed collection has been conserved at the Germplasm Bank of the Universidad Politécnica de Madrid. However, as some seed samples had not been labelled clearly at the time of collection, their origin was uncertain. METHODS: Genetic variation in germplasm accessions of B. chouardii was investigated using microsatellite (simple sequence repeat; SSR) markers. KEY RESULTS: The 17 primer pairs used detected 62 SSR alleles in the 46 samples analysed from five different germplasm stocks. Eight alleles scored from the wild population were not detected in the germplasm samples analysed. The relatedness of the germplasm samples to the wild subpopulations through neighbour-joining clustering, principal coordinates analysis (PCO) and assignment tests revealed a biased higher representation of the genetic diversity of the lower cliff (43 samples) subpopulation than that of the upper cliff (three samples). CONCLUSIONS: The collection of additional samples from the upper cliff is recommended to achieve a better representation of the genetic diversity of this subpopulation. It is also recommended that these stocks should be managed separately according to their distinct microspatial origin in order to preserve the genetic substructuring of the wild population.     

Genetic monitoring as a promising tool for conservation and management

In response to ever-increasing anthropogenic changes to natural ecosystems, regional, national and international organizations have established guidelines for monitoring biological diversity. Most monitoring programs, however, do not take full advantage of the potential afforded by molecular genetic markers, which can provide information relevant to both ecological and evolutionary time frames, while costing less and being more sensitive and reliable than traditional monitoring approaches. As several molecular and computational approaches are relatively new, many technical and theoretical issues remain to be resolved. Here, we illustrate how DNA and population genetic data can provide valuable information, often unattainable via other approaches, for monitoring species of management, conservation and ecological interest.     

The evolution of sexual reproduction as a repair mechanism. Part I. A model for self-repair and its biological implications

Summary The theory is presented that the sexual process is a repair mechanism which maintains redundancy within the sub-structure of hierarchical, self-reproducing organisms. In order to keep the problems within mathematically tractable limits (see Part II), a simple model is introduced: a wheel with 6 spokes, 3 of them vital and 3 redundant, symbolizes the individual (cell or organism). Random accidents destroy spokes; the wheels replicate at regular cycles and engage periodically in pairing and repair phases during which missing spokes are copy-reproduced along the intact spokes of the partner wheel.The hierarchical structure of such a system is analysed and an autonomous unit is defined: this is the unit of minimal hierarchical complexity which is capable of perpetuating autonomously all higher and all lower levels of the hierarchy; this is the central unit of selection.Four basic, physical parameters are isolated which determine the essential features of any eucaryotic life cycle: 1. The number of levels of the hierarchy (unicellular, multicellular, colonial, etc.); 2. the relation between the phases of replication (asexual generations) and repair (sexual generations); 3. the duration of potential repair (haplo- diplo-phases); 4. the position of the sexual partners within the hierarchy (selfing, monecy, dioecy, reproductive individuals within colonies, etc.).The evaluation of fitness components is considered in relation to trends of reproductive patterns in evolution.     

Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model

Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine residues. The procedure delivers two sperm fractions: annexin V-negative (nonapoptotic) and annexin V-positive (apoptotic). Our aim was to determine whether the sperm fertilizing potential can be improved by selecting a nonapoptotic fraction using MACS. Semen samples (n = 35) were subjected to separation on a density gradient followed by MACS. Extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labeled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and DNA fragmentation using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay. The sperm fertilization potential was assessed using hamster oocyte penetration assay and hamster oocyte-intracytoplasmic sperm injection (ICSI). Annexin V-negative sperm displayed superior quality in terms of high motility, low caspase 3 activation, MMP integrity, and small extent of DNA fragmentation. Annexin V-negative sperm demonstrated higher oocyte penetration capacity but comparable sperm chromatin decondensation (SCD) following ICSI. Conversely, the annexin V-positive sperm presented with poor quality and fertilization potential. The oocyte penetration rate was negatively correlated with apoptotic marker expression, whereas SCD following ICSI was only associated with apoptosis on sperm-damaged membranes. We conclude that apoptosis appears to impact sperm-oocyte penetration rate; however, it does not seem to affect early stages of fertilization such as SCD in spermatozoa of healthy donors. The selection of nonapoptotic sperm by MACS may be used to enhance results of in vitro fertilization by increasing sperm-oocyte penetration.     

Novel micropropagation and weaning methods for the integrated conservation of a critically endangered tree species, Medusagyne oppositifolia

Medusagyne oppositifolia is a critically endangered monotypic species with only a few trees left in the wild. Due to invasion and habitat clearance, genetic diversity of this species has been reduced to an alarmingly low level. Long-term in vitro cultures of M. oppositifolia were slow-growing and eventually lost the ability to multiply and root. Because of the lack of new seed and vegetative materials, efforts were made to rejuvenate the existing cultures. This was attempted by a series of steps involving serial pruning of the shoot tips and growing on a modified MS medium containing silver nitrate and thidiazuron. Rapid cycling clonal propagules were produced this way from cultured nodes. Isolated shoots were rooted on Florialite?, and rooted plants were weaned in a transition stage using a Nutrient Film Technique (NFT) hydroponic system before being completely weaned under glasshouse conditions. The importance of screening cultures for multiplication and rooting efficiency during long-term maintenance is discussed in the context of stress metabolite production and its influence on growth in culture. Factors contributing to rejuvenation of old cultures, application of unconventional supporting systems for rooting and application of the hydroponic system as a weaning tool are also discussed.     

Proteomics as a tool for the investigation of seafood and other marine products

The state-of-the-art and future trends of the application of proteomics to seafood and other marine products are reviewed. Consumers' demands for seafood products have increased in the recent years and this situation has underlined the need to guarantee the safety, traceability, authenticity, and health benefits of such products. The increasing presence of commercially available aquaculture products has also prompted the seafood industry to face newer challenges. In this sense, a review of the present status and perspectives of the application of proteomics in the development of newer biotechnology products of marine origin is given.     

Minimalist protein model as a diagnostic tool for misfolding and aggregation

We propose a realistic coarse-grained protein model and a technique to "anchor" the model to available experimental data. We apply this procedure to characterize the effect of multiple mutations on the folding mechanism of protein S6. We show that the mutation of a few "gatekeeper" residues triggers significant changes on the folding landscape of S6. These results suggest that gatekeeper residues control the flexibility of critical regions of S6, that in turn regulates the delicate balance between folding and aggregation. Although obtained with a minimalist protein model, these results are fully consistent with experimental evidence and offer a clue to understand the interplay between folding and aggregation in protein S6.     

The Iberian Peninsula as a potential source for the plant species pool in Germany under projected climate change

The application of niche-based modelling techniques to plant species has not been explored for the majority of taxa in Europe, primarily due to the lack of adequate distributional data. However, it is of crucial importance for conservation adaptation decisions to assess and quantify the likely pool of species capable of colonising a particular region under altered future climate conditions. We here present a novel method that combines the species pool concept and information about shifts in analogous multidimensional climate space. This allows us to identify regions in Europe with a current climate which is similar to that projected for future time periods in Germany. We compared the extent and spatial location of climatically analogous European regions for three projected greenhouse gas emission scenarios in Germany for the time period 2071–2080 (+2.4°C, +3.3°C, +4.5°C average increase in mean annual temperature) to those of the recent past in Europe (1961–90). Across all three scenarios, European land areas which are characterised by climatic conditions analogue to those found in Germany decreased from 14% in 1961–1990 to ca. 10% in 2071–2080. All scenarios show disappearing current climate types in Germany, which can mainly be explained with a general northwards shift of climatically analogous regions. We estimated the size of the potential species pool of these analogous regions using floristic inventory data for the Iberian Peninsula as 2,354 plant species. The identified species pool in Germany indicates a change towards warmth and drought adapted southern species. About one-third of the species from the Iberian analogous regions are currently already present in Germany. Depending on the scenario used, 1,372 (+2.4°C average change of mean annual temperature), 1,399 (+3.3°C) and 1,444 (+4.5°C) species currently not found in Germany, occur in Iberian regions which are climatically analogous to German 2071–80 climate types. We believe that our study presents a useful approach to illustrate and quantify the potential size and spatial distribution of a pool of species potentially colonising new areas under changing climatic conditions.     

The mouse as a model for human biology: a resource guide for complex trait analysis

The mouse has been a powerful force in elucidating the genetic basis of human physiology and pathophysiology. From its beginnings as the model organism for cancer research and transplantation biology to the present, when dissection of the genetic basis of complex disease is at the forefront of genomics research, an enormous and remarkable mouse resource infrastructure has accumulated. This review summarizes those resources and provides practical guidelines for their use, particularly in the analysis of quantitative traits.     

Implications of seed germination ecology for conservation of Camptotheca acuminata,a rare,endemic, and endangered species in China

Plant Ecology - Seed germination and seedling recruitment are among the most critical stages for plant population persistence and development, which may be influenced by habitat fragmentation and...     

Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species

The survival of hundreds of threatened amphibian species is increasingly dependent on conservation breeding programs (CBPs). However, there is an ongoing loss of genetic variation in CBPs for most amphibians, reptiles, birds, and mammals. Low genetic variation results in the failure of CBPs to provide genetically competent individuals for release in supplementation or rehabitation programs. In contrast, in the aquaculture of fish the perpetuation of genetic variation and the production of large numbers of genetically competent individuals for release is accomplished through the cryopreservation of sperm. Successful protocols for the cryopreservation of amphibian sperm from excised testes, and the use of motile frozen then thawed sperm for fertilisation, have been adapted from those used with fish. However, there have been no protocols published for the cryopreservation of amphibian hormonally induced sperm (HIS) that have achieved fertility. We investigated protocols for the cryopreservation of amphibian HIS with the European common frog (Rana temporaria) as a model research species. We induced spermiation in R. temporaria through the intraperitoneal administration of 50 μg LHRHa and sampled HIS through expression in spermic urine. Highly motile HIS at a concentration of 200 × 10⁶/mL was then mixed 1:1 with cryodiluents to form cryosuspensions. Initial studies showed that; 1) concentrations of ∼15 × 10⁶/mL of HIS achieve maximum fertilisation, 2) TRIS buffer in cryodiluents did not improve the recovery of sperm after cryopreservation, and 3) high concentrations of DMSO (dimethylsulphoxide) cryoprotectant reduce egg and larval survival. We then compared four optimised cryopreservation protocols for HIS with the final concentrations of cryodiluents in cryosuspensions of; 1) DMSO, (½ Ringer Solution (RS), 10% sucrose, 12% DMSO); 2) DMSO/egg yolk, (½ RS, 10% sucrose, 12% DMSO, 10% egg yolk), 3) DMFA, (½ RS, 10% sucrose, 12% dimethylformamide (DMFA)), and 4) MIS/glycerol, (Motility Inhibiting Saline (MIS), 5% glycerol, 2.5% sucrose, 5% egg yolk). Cryosuspensions were frozen in LN₂ vapour, stored in LN₂, thawed in 40° C water bath, and activated by slow equilibration with 1:3 dilutions of cryosuspensions with 20 mM/L NaCl. Protocol efficacies were assessed through the post-thaw percentage of; 1) sperm motility, 2) sperm membrane integrity, 3) fertilisation, 4) fertilised eggs hatching, and 5) larval survival from fertilised eggs to 7 d. The DMFA cryodiluent proved superior to the DMSO based cryodiluents in recovery of sperm motility and fertility after cryopreservation. MIS/glycerol cryodiluent provided low sperm viability and no fertility. Considering the ease of obtaining HIS from many Rana species, the success of our protocols offer the potential for the perpetuation of the genetic variation of the 42 threatened Rana species and the 193 threatened Ranid species in total.