Ovarian control for assisted reproduction in the domestic cat and wild felids

Of the 37 felid species, all but the domestic cat are classified as threatened with extinction in all or part of their native range. Additionally, the domestic cat is a valuable model for human biomedical research. Propagating some wild felids as well as domestic cat populations serving as human models is a major challenge primarily due to difficulties in transporting animals between facilities to ensure the pairing of genetically matched individuals, behavioral incompatibility between pairs and low fertility. Artificial insemination (AI) and in vitro fertilization/embryo transfer (IVF/ET) are powerful tools for helping manage rare populations. Developing successful assisted reproductive techniques requires knowledge of the female reproductive cycle and precise control of ovarian activity. Successful ovarian stimulation for AI and IVF/ET has been achieved in at least one-third of all cat species. However, sensitivity to a given gonadotropin treatment appears highly species-specific, and poor responses are common, particularly in felid species that exhibit spontaneous ovulations. Furthermore, current gonadotropin regimens have been demonstrated to perturb female reproductive function often leading to reduced fertility. Overall, ovarian response to exogenous hormonal stimulation has been highly variable, and pregnancy success after AI or IVF/ET remains low (<20%) in most species. Therefore, there is an immediate need to develop improved regimens that would allow more predictable ovarian responses in felids. We contend that recent research involving the use of progestins to control the ovary prior to gonadotropin stimulation shows promise for providing consistent ovarian stimulation in felids.     

Current progress on assisted reproduction in dogs and cats: in vitro embryo production

The objective of the development of assisted reproduction techniques in dogs and cats is their application to non-domestic canine and feline species, most of which are considered threatened or endangered. Among these techniques, an entirely in vitro system for embryo production is effectively an important tool for conservation of wildlife. In the last decade, progress has been made in embryo production in carnivores. It has been shown that canine oocytes can resume meiosis in vitro and that these oocytes can be fertilized and developed in vitro, although at a much lower rate than most other domestic animal oocytes. The reason lies in the dissimilarities of reproductive physiology of the dog compared to other species and the lack of precise information concerning the oviductal environment, in which oocyte maturation, fertilization and early embryonic development take place. Successful in vitro embryo production in the domestic cat has been attained with oocytes matured in vitro, and kittens were born after transfer of IVM/IVF derived embryos. On the basis of these results the in vitro fertilization of oocytes has also been applied in several non-domestic feline species. The effectiveness of such protocols in the preservation of genetic material of rare species can be improved by developing better techniques for long-term storage of gametes. In dogs and cats sperm cells have been successfully frozen and the cryopreservation of oocytes would greatly increase their availability for a range of reproductive technologies. Cryopreserved cat oocytes can be fertilized successfully and their development in vitro after fertilization is enhanced when mature oocytes are frozen. Thus refined techniques of oocyte maturation and fertilization in vitro coupled with oocyte cryopreservation could allow for an easy establishment of genetic combinations when male and female gametes in the desired combination are not simultaneously available, and the propagation of endangered carnivores would be facilitated.     

Research in nondomestic species: experiences in reproductive physiology research for conservation of endangered felids

Tremendous strides have been made in recent years to broaden our understanding of reproductive processes in nondomestic felid species and further our capacity to use this basic knowledge to control and manipulate reproduction of endangered cats. Much of that progress has culminated from detailed scientific studies conducted in nontraditional laboratory settings, frequently at collaborating zoological parks but also under more primitive conditions, including in the field. A mobile laboratory approach is described, which incorporates a diverse array of disciplines and research techniques. This approach has been extremely useful, especially for conducting gamete characterization and function studies as well as reproductive surveys, and for facilitating the development of assisted reproductive technology. With continuing advances in assisted reproduction in rare felids, more procedures are being conducted primarily as service-related activities, targeted to increase effectiveness of species propagation and population management. It can be a challenge for both investigators and institutional animal care and use committees (IACUCs) to differentiate these service-based procedures from traditional research studies (that require IACUC oversight). For research with rare cat species, multi-institutional collaboration frequently is necessary to gain access to scientifically meaningful numbers of study subjects. Similarly, for service-based efforts, the ability to perform reproductive procedures across institutions under nonstandard laboratory conditions is critical to applying reproductive sciences for managing and preserving threatened cat populations. Reproductive sciences can most effectively assist population management programs (e.g., Species Survival Plans) in addressing conservation priorities if these research and service-related procedures can be conducted "on the road" at distant national and international locales. This mobile laboratory approach has applications beyond endangered species research, notably for other scientific fields (e.g., studies of hereditary disease in domestic cat models) in which bringing the laboratory to the subject is of value.     

Genetic diversity and conservation in a small endangered horse population

The Old Kladruber horses arose in the 17th century as a breed used for ceremonial purposes. Currently, grey and black coat colour varieties exist as two sub-populations with different recent breeding history. As the population underwent historical bottlenecks and intensive inbreeding, loss of genetic variation is considered as the major threat. Therefore, genetic diversity in neutral and non-neutral molecular markers was examined in the current nucleus population. Fifty microsatellites, 13 single nucleotide polymorphisms (SNPs) in immunity-related genes, three mutations in coat colour genes and one major histocompatibility (MHC-DRA) gene were studied for assessing genetic diversity after 15 years of conservation. The results were compared to values obtained in a similar study 13 years ago. The extent of genetic diversity of the current population was comparable to other breeds, despite its small size and isolation. The comparison between 1997 and 2010 did not show differences in the extent of genetic diversity and no loss of allele richness and/or heterozygosity was observed. Genetic differences identified between the black and grey sub-populations observed 13 years ago persisted. Deviations from the Hardy–Weinberg equilibrium found in 19 microsatellite loci and in five SNP loci are probably due to selective breeding. No differences between neutral and immunity-related markers were found. No changes in the frequencies of markers associated with two diseases, melanoma and insect bite hypersensitivity, were observed, due probably to the short interval of time between comparisons. It, thus, seems that, despite its small size, previous bottlenecks and inbreeding, the molecular variation of Old Kladruber horses is comparable to other horse breeds and that the current breeding policy does not compromise genetic variation of this endangered population.     

Spatial variation in population dynamics of juvenile Atlantic salmon: implications for conservation and management

Application of habitat models for predicting expected local densities of Atlantic Salmon Salmo salar in healthy populations has been hampered by a lack of generality in their fit to data from different systems. It is believed that this problem results at least in part from difficulties of effectively integrating factors that act across a range of spatial and temporal scales. Here, as an aid to developing more robust modelling and sampling methodologies, a simple process‐based model for local‐scale dynamics of Atlantic salmon juveniles is developed from first principles by integrating contemporary understanding of self‐thinning, density‐dependent growth and dispersal. The aim is to present a readily understood structure to illustrate the links between spawning and stocking strategies, habitat, migration and fish production. Based on this structure, contemporary understanding of the more complex biological processes that affect density, growth and habitat are discussed in relation to some of the key requirements of managers, including stocking for rehabilitation, assessment of predation impact and development of strategies for sampling populations effectively when deriving habitat‐production models. A major conclusion is that more structured, integrated research is required to provide the basic variables needed to model links between local and global scale habitat and fish production effectively. Nevertheless, application of the current understanding of the biology of Atlantic salmon should be of great benefit to managers in extracting key information from field surveys.     

The potential of certification for conservation and management of wild MAP resources

Medicinal and aromatic plants (MAPs) are an integral part of our biodiversity. In majority of MAP rich countries, wild collection practices are the livelihood options for a large number of rural peoples and MAPs play a significant role in socio-economic development of their communities. Recent concern over the alarming situation of the status of wild MAP resources, raw material quality, as well as social exploitation of rural communities, leads to the idea of certification for MAP resource conservation and management. On one hand, while MAP certification addresses environmental, social and economic perspectives of MAP resources, on the other hand, it ensures multi-stakeholder participation in improvement of the MAP sector. This paper presents an overview of MAP certification encompassing its different parameters, current scenario (Indian background), implementation strategies as well as stakeholders’ role in MAP conservation. It also highlights Indian initiatives in this direction.     

A comparison of management strategies for conservation with regard to population fitness

Computer simulations have been carried out tocompare, under realistic genetic models, twomethods proposed in the literature to retaingenetic diversity in conservation programmes.In a two-step method, contributions of parentsare set up to produce minimum coancestry(kinship) among the offspring, and this isindependent of the mating system subsequentlyapplied. In a single-step method,contributions and matings are decidedsimultaneously in order to minimise coancestry.The comparison is made in terms of maintainedgenetic diversity and in terms of populationfitness. We conclude that the two methodsmaintain approximately the same geneticdiversity but the latter induces higher levelsof inbreeding, reducing the fitness of thepopulation. Avoidance of close relatives'matings improves this latter method, but thefitness levels do not reach those of thetwo-step scheme. We also investigate theperformances of different mating strategies incombination with minimum coancestry (two-stepmethod), concluding that these mating systemsdo not substantially affect the effectivenessof the management. Finally, we illustrate howminimum group coancestry can be restrictedto a minimum loss of fitness, if a measure ofthis is available for the individuals.     

Strong population genetic structure and its implications for the conservation and management of the endangered Itasenpara bitterling

To infer the population genetic structure and genetic diversity of Itasenpara bitterling (Acheilognathus longipinnis), a cyprinid species endemic to Japan and distributed in only three specific regions, we investigated mitochondrial DNA variation. The distribution of the haplotypes among the three regions showed distinct geographic structure, and no common haplotypes were observed among regions. Analysis of molecular variance revealed a significant proportion of the genetic variance was partitioned among regions (93.1 %, P < 0.001), and pairwise estimates of D_A and Φ_ST between regions also revealed strong population structure. Given the strong genetic structure and low genetic diversity within regions, we strongly suggest that each region should be treated as a separate unit in any conservation program and any inter-regional translocations should be avoided.     

Hope springs eternal in the starfish gonad: preserved potential for sexual reproduction in a single-clone population of a fissiparous starfish

Among echinoderms, asexual reproduction by fission occurs in few species. This strategy is considered a temporary response to stressful conditions and usually alternates with sexual reproduction events; thus, monoclonal populations are extremely rare. The occurrence of a single-clone population of the starfish Coscinasterias tenuispina at Llançà (NW Mediterranean) allowed us to study intra-clonal variation of the reproductive cycle during a two-year study. The few developed gonads (all male) were found in winter months, coinciding with the minimum photoperiod (ρ = ?0.82; P < 0.001) and lowest temperatures (ρ = ?0.75; P < 0.001), only in best-fed individuals, indicating that food availability influences individual ability for gonad development. Fissiparity happened throughout all the sampled period, but its rate increased with warm temperatures (ρ = 0.68; P < 0.0001). In contrast to what has been reported in other species, no correlation between fission rates and population density was found. The population was maintained over time by asexual reproduction and remained monoclonal. Although sexual reproduction has probably not occurred in this all-male population for a long time, the ability to yearly produce mature gonads is retained by some individuals, indicating that potential to reproduce sexually may be preserved, even in the case of strictly asexual populations.     

Production of embryos by assisted reproduction in the horse

In vitro embryo production is not yet successful in the horse, largely due to low rates of fertilization in vitro. However, methods to produce embryos from isolated oocytes have been developed. Oocytes may be recovered from living mares by aspiration of the dominant preovulatory follicle by trans-abdominal puncture, and from both preovulatory and immature follicles by trans-vaginal ultrasound-guided puncture. Transfer of in vivo-matured oocytes to the oviducts of bred recipient mares has resulted in good pregnancy rates (75-85%). Little work has been done on transfer of horse oocytes matured in vitro. Recovery rates of immature oocytes from mares in vivo are lower than those for cattle. In addition, work on oocytes recovered from horse ovaries post-mortem has shown that horse oocytes from smaller (< 20 mm diameter) viable follicles may not yet be meiotically competent. Methods for in vitro fertilization and for obtaining adequate numbers of competent immature oocytes from the mare must be developed before in vitro embryo production can become a useful clinical and research procedure in the horse.     

Predator abundance in relation to small game management in southern Portugal: conservation implications

The interaction between hunting interests and legally protected predators is often a contentious conservation problem, requiring detailed understanding of predator responses to game management. This issue was addressed in southern Portugal in a treatment-control natural experiment, whereby the abundances of small game, corvids, birds of prey and carnivores were compared in 12 game estates (>500 ha) and 12 matching areas with similar sizes and land uses but no game management. European rabbits (Oryctolagus cuniculus), Iberian hares (Lepus granatensis) and, less so, red-legged partridges (Alectoris rufa) were far more numerous in game estates than elsewhere. Among legally controlled species, there were less Eurasian jays (Garrulus glandarius) but more red foxes (Vulpes vulpes) in game estates, though the latter were primary targets of predator culling. Fox abundance within game estates varied inversely with an index of management intensity (density of small game feeding sites) and increased along with hare abundance. As for protected species, only common kestrels (Falco tinnunculus) and genets (Genetta genetta) were fewer in game estates. The abundance of raptors within game estates varied inversely with gamekeeper density, whereas that of common buzzards (Buteo buteo) increased along with rabbit abundance. Overall, there was little evidence that game management reduced local predator abundances, except in the most intensively managed estates. Game estates provided concentrations of prey that was scarce elsewhere, which may have favoured increased abundances of some predators. Further investigations are needed to find out whether high prey densities may attract predators to game estates with increased mortality risk, which may thus become population sinks for protected species.     

Toward culture of single gametes: the development of microfluidic platforms for assisted reproduction

During the last few decades in vitro production of mammalian embryos and assisted reproductive technologies such as embryo transfer, cryopreservation, and cloning have been used to produce and propagate genetically superior livestock. However, efficiencies of these technologies remain low. For these technologies to become more commercially viable, the efficiencies must improve. Despite this importance of reproduction for the livestock industry, little progress in decreasing embryonic mortality has been made. The livestock industry has succeeded in achieving large increases in average milk production of dairy cattle, growth rate in beef cattle and leanness in swine but reproductive efficiency has actually decreased. For example, research has provided little progress toward developing an objective method to examine viability of a single living embryo. At the same time, the growth of miniaturization technologies beyond integrated circuits and toward small mechanical systems has created opportunities for fresh examination of a wide range of existing problems. While the investigation and application of miniaturization technologies to medicine and biology is progressing rapidly, there has been limited exploration of microfabricated systems in the area of embryo production. Microfluidics is an emerging technology that allows a fresh examination of the way assisted reproduction is performed. Here we review the progress in demonstrating microfluidic systems for in vitro embryo production (IVP) and embryo manipulation. Microfluidic technology could have a dramatic impact on the development of new techniques as well as on our basic understanding of gamete and embryo physiology.     

Food provision to nestlings in the Hoopoe Upupa epops: implications for the conservation of a small endangered population in the Swiss Alps

In an attempt to recognize the possible ecological causes of the decline of a population of Hoopoes Upupa epops in the Swiss Alps, we collected data on resource exploitation. The prey provisioned to nestlings by parents was investigated at four breeding sites using photographs (n = 4353, 80% of which enabled prey identification). Molecrickets Gryllotalpa gryllotalpa and Lepidoptera (larvae and pupae) were dominant in nestling diet (93% frequency; 97% biomass). Although Molecrickets were provisioned less frequently (26%) than Lepidoptera (67%), they represented 68% of the total biomass (vs 29% for Lepidoptera). There was an overall negative relationship between the proportion of Molecricket biomass in the diet and the parents' feeding rate, whereas a comparison between broods showed that a higher provisioning activity did not lead to an increase in the biomass supplied to the chicks. A diet based on Molecrickets therefore appears to be energetically advantageous. As Molecrickets are a traditional prey of Hoopoes in central Europe, this might be relevant to other populations. In the study area, Molecrickets occur only on the intensively cultivated plain, whereas the majority of Hoopoe pairs nest at various altitudes on the foothills adjacent to the plain as the latter provides at present almost no suitable nesting sites. Hoopoes breeding higher up on the foothills seem to experience greater provisioning costs and have, on average, lower breeding success. Providing nest sites on the plain is the main conservation measure proposed for the local Hoopoe population. Further attention should also be paid to Molecrickets as these may be crucial for Hoopoes.     

Mitochondrial DNA variations in ova and blastocyst: Implications in assisted reproduction

Mitochondrial DNA (mtDNA) of oocyte is critical for its function, embryo quality and development. Analysis of complete mtDNA of 49 oocytes and 18 blastocysts from 67 females opting for IVF revealed 437 nucleotide variations. 40.29% samples had either disease associated or non-synonymous novel or pathogenic mutation in evolutionarily conserved regions. Samples with disease associated mtDNA mutations had low fertilization rate and poor embryo quality, however no difference in implantation or clinical pregnancy rate was observed. Screening mtDNA from oocyte/blastocyst is a simple, clinically reliable method for diagnostic evaluation of female infertility and may reduce risk of mtDNA disease transmission.