Current research projects

01_Reproduction biology of lynx – basic research for conservation breeding of Iberian lynx

The Iberian lynx (Lynx pardinus Temminck, 1827), a specialist predator of wild rabbits, exclusively found on the Iberian Peninsula, was declared as critically endangered (IUCN 2002). As a consequence, an integrated species conservation plan which links in situ conservation efforts with a conservation breeding program was started to prevent the Iberian lynx from extinction. Researchers of the Leibniz-Institute for Zoo- and Wildlife Research (IZW) are scientific partners of the Iberian lynx conservation program since 2005.

Within this long-term project we not only successfully established a new method for non-invasive pregnancy diagnosis in feline species and validated an assay for faecal glucocorticoid determination in lynxes but also elucidated a unique reproductive peculiarity of lynxes in comparison to other felids. Lynxes develop physiologically persistent corpora lutea (CL) which preclude common approaches of estrous management to exploit multiple cycles of a female per season for assisted reproduction.

contact: Prof. Katarina Jewgenow, Dr. Beate Braun

 

 

02_Biobanking – field friendly cryopreservation

Biobanking requires the maintenance of functional cell properties and the protection from microbial contaminants. According to the unique opportunities of IZW to get access to gonads of rare or endangered animals we continuously perform biobanking of gametes and reproductive tissues. However, the physiological function of cells and tissue is usually compromised by the cryopreservation process. Therefore we elucidate the impact of assisted reproductive techniques (ART) and refine our methods to maintain functionality. For instance, we defined an impact of sperm lipid composition on species-specific freezing ability, and examined a protective potential of selected lipid supplements or seminal fluid components.  We developed field-suited protocols, reproducible by non-skilled persons, for cryopreservation of felid and other mammalian sperm and ovary tissue. Recently, we developed a protocol for cryopreservation of dissociated cells from felid testes. The IZW cryobank biosafety was confirmed in a ring-study guided by the Association of German Cryobanks (GDK) where microbial contaminations were comprehensively tested.

contact: Dr. Karin Müller, Dr. Jennifer Zahmel, Prof. Katarina Jewgenow

 

 

03_Non-invasive pregnancy diagnosis in carnivores

 

The gold standard for pregnancy diagnosis is ultrasound confirmation. Practically, this is generally unfeasible without physical or chemical restraint in most zoo- and wildlife species. An alternative way to predict, or tentatively confirm, ongoing pregnancy, is non-invasive endocrine monitoring. This is fairly easy in some species (e.g. progesterone-metabolite analysis in elephants), but remains extremely challenging in other species, particularly those showing delayed implantation and pseudopregnancy, amongst them canine, feline and bear species. In this project we aim to identify biomarkers for pregnancy, based on several technologies including immunological assays and state-of-the art  (U)HPLC-MS.

contact: Dr. Jella Wauters

 

04_Felid-Gamete-Rescue-Projekt

Due to continuous decline of felid populations in the wild many felid species are listed in different categories on the IUCN Red List of threatened species. Main reasons for declining numbers are habitat fragmentation and loss or illegal hunting for meat, traditional chinese medicine or trophy souvenirs.

With the decline in the wild, felid populations in zoos gain increasing relevance as back-up populations. By now the replenishment of zoo populations with animals from the wild for blood replacement is unrealistic for most felid species.

Zoos have to manage their animals carefully and globally to safe the genetic diversity and to maintain healthiness of their small fragmented felid populations. Therefore, 27 European studbooks and European Endangered Species Programs exist for felid species or subspecies.

The department “reproduction biology” in cooperation with the departments “wildlife diseases” and “reproduction management” seeks to provide a research-based. enhancement to the zoos efforts and therefore established a gamete bank for all felid species (named “Felid-Gamete-Rescue-Project”) with two aims:

-  All non-established methods of assisted reproductive technologies (e.g. in-vitro fertilization of oocytes (IVF), in-vitro culture of embryos, embryo transfer, cryopreservation of gonadal tissue, sperm, oocytes and embryos) will be investigated and continuously adapted and improved.

- Zoos are requested to place sperm, oocytes and tissue of their euthanized or castrated felids into stock but can also request material for artificial inseminations or embryo transfer. This would allow bringing genetic material of already deceased animals back into the existing populations. In the course of the last 10 years, we have received samples of 141 different individuals offered by 36 different zoos. 1110 oocytes have been cultured and 47 embryos were produced and cryopreserved. One highlight was the generation of an Asiatic Golden Cat embryo, a felid species with less than 20 individuals left in European zoos. Sperm samples of 36 different males were cryopreserved and a field-friendly method of sperm cryopreservation has been developed and published.

contact: Dr. Jennifer Zahmel

 

05_Luteal function in lynx species

Lynxes show the reproductive peculiarity of persistent corpora lutea (CL). We comprehensively characterized lynx CL in comparison to cat CL with the aim to use this knowledge for estrous management of Iberian lynx and other feline species. Molecular expression patterns were compared between persistent lynx CL and life cycle stages of CL from domestic cat to identify factors which are responsible for CL persistency. Beside prostaglandin E (PGE), we recognized estrogen and prolactin as potential luteotrophic factors.

Because of the restricted access to wildlife  animals we successfully established protocols for short-term luteal cell cultures to further investigate luteal life cycle regulation in feline species and to develop protocols for artificial luteolysis of persistent CL.

contact: Dr. Beate Braun, Prof. Katarina Jewgenow

 

 

06_Determination of steroid hormones in hair samples

Non-invasive monitoring allows hormone analysis of zoo-and wildlife without disturbance. Best explored matrices are f faeces and urine, but their (fresh) sampling is sometimes challenging, particularly in wild ranging individuals. An elegant alternative to determine the endocrine status would be through hair analysis. In this project we are investigating the suitability of hair as a matrix to study the reproductive status (e.g. progesterone as a measure of pregnancy) or well-being (e.g. glucocorticoids as a measure of stress) of an individual. Additionally, hair sex steroid measurement may perhaps allow sex-determination in wild ranging individuals.

Hair analysis is challenged at many levels, and proper optimization and validation of the extraction and detection (EIA) techniques are mandatory for each studied species. For this purpose, HPLC and (U)HPLC-MS/MS are being explored to confirm the validity of our developed extraction and EIA methods.

contact: Prof. Katarina Jewgenow, Dr. Jella Wauters

 

 

07_Key factors of gonadal development

The identification of regulatory key molecules of feline spermatogenesis or early folliculogenesis allows us to assess sexual maturation or seasonal reproductive changes in individuals. Moreover, it helps to identify suitable markers for testicular and ovary cell types and stages which are not yet available in felids. These can be used for the characterization and improvement of in vitro cultivation of testicular and ovarian tissue.

contact: Dr. Beate Braun

 

08_Seminal fluid

Seminal fluid is missing in sperm samples recovered from epididymides of valuable dead or castrated males, and is highly diluted in cryopreserved semen samples. We aim to decipher selected seminal fluid components (e. g. antioxidative system, spermadhesins) which improve fertilization efficiency if supplemented to gamete samples in artificial reproductive techniques (ART). For example, a high antioxidative capacity in lion seminal fluid protects sperm better from cryodamage.

Contact: Dr. Karin Müller, Dr. Beate Braun

 

09_Lipid function in semen

The contribution of lipids as structural and functional membrane components for fertilization competence and cryotolerance of male gametes is under investigation. The protection of membrane lipids against oxidation and semen lipid repair capacity is species specific. One consequence is for instance to develop species specific extender supplements like e. g. combinations of selected fatty acids than to use common native lipid supplements such as egg yolk.

contact: Dr. Karin Müller

 

10_Cellular basis of gamete-reproductive tract interactions

Understanding of gamete-reproductive tract interactions is a precondition to improve the outcome of assisted reproduction techniques. Options for in vivo experiments are limited for ethical and practical reasons. Establishing cell culture models (e. g. oviduct epithelial cells) to study reproductive functions in vitro is a powerful tool to generate missing knowledge (control of ovary activity, oviduct interaction with preserved sperm cells prior fertilization).

Examples: Seminal fluid which is missing or highly diluted in most cryobanked feline sperm samples increases the number of oviduct-bound sperm in vitro. The procedure of cryopreservation impairs this interaction.

contact: Dr. Karin Müller

 

11_Signatures of fe-/male fertility - Using long-term selection for high fertility to decipher the genetics of increased reproductive performance

By comparing genomes of high fertility mouse lines with genomes of non-selected mice we aim to identify signatures of selection (= occurrence and frequency patterns of alleles causal for the selected reproductive trait). General applicability of results will then be tested in other mammal species (pigs, lions) by FBN Dummerstorf and IZW, Dept. of Evolutionary Genetics. Sperm parameters to correlate male fertility will be evaluated (IZW, Dept. Reproduction Biology, IFN Schönow).

contact: Dr.Karin Müller

 

12_Paternal epigenetic effects

Heritable epigenetic changes, or transgenerational effects, are the result of the fixation of epigenetic markers in the genome of gametes as a result of environmental impacts. Past research was focused on maternal epigenetics, neglecting the paternal influence on the next generation. To identify paternal effects we test in the wild guinea pig whether alterations of environmental conditions lead to changes in the methylation patterns in fathers. We also investigate whether those changes are also detectable in the male offspring, and thus paternally heritable.

contact:Prof. Katarina Jewgenow, Department 2 projects