Biobanking for assisted reproduction techniques

Assisted reproduction techniques help to maintain the biodiversity. In particular the cryopreservation of gametes is an essential option to preserve the genetic diversity of wild animals and to support breeding programs in zoos.

Project details
Duration: since 05/2007
Third-party funded: partly
Involved Department(s): Dept Reproduction Biology, Dept Reproduction ManagementDept Wildlife Diseases
Leibniz-IZW Project Leader(s): Karin Müller, Jennifer Zahmel (all: Dept Reproduction Biology)
Leibniz-IZW Project Team:

Katarina Jewgenow (Dept Reproduction Biology), Gudrun Wibbelt, Claudia Szentiks (all: Dept Wildlife Diseases), Frank Göritz (Dept Reproduction Management)

Lorena Fernández-González (since 2013), Mohammad Bashawat (since 2017), Susanne Eder (2012-2020), Martina Colombo (2019-2020) (all: Dept Reproduction Biology)
Consortium Partner(s): Alliance of German Cryobanks (Gemeinschaft Deutscher Kryobanken, GDK)
Current Funding Organisation: Minitüb GmbH
Research Foci:

Improving population viability

Development of theory, methods and tools


Assisted reproduction techniques help to maintain biodiversity. We aim to continuously improve the protocols for a long-term preservation of gametes and reproductive organ tissues of a variety of species at deep temperatures. In 2007, we established a specific gamete bank for felid species to support breeding programs in zoos. To enable a comprehensive application of the preserved cell and tissue samples we investigate and optimize a variety of in vitro methods of assisted reproduction.

M. Colombo et al., talk at the CRYO2020 (57th Annual Meeting of the Society for Cryobiology)

Project focus: Biobanking

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) such as cryopreservation and refine our methods to maintain the functionality of cells and tissues. 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 certified in a ring-study guided by the Association of German Cryobanks (GDK) where microbial contaminations were comprehensively tested. We closely collaborate with the colleagues of Dept Wildlife Diseases (pathology) to ensure the optimal rescue of samples, as well as with the veterinarians of Dept Reproduction Management.


 I am Dr. Mohammad Bashawat, Postdoc in Department 4. If you would like to know more about my project, please watch the video!

Project focus: Felid Gamete Rescue Project

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 to recharge genetic variety is unrealistic for most felid species.

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

We aim to provide research-based enhancement to the zoos’ efforts and therefore established a gamete bank for all felid species (named “Felid-Gamete-Rescue-Project”) with two goals:

  • All methods of assisted reproductive technologies not yet established in felids (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 zoos. 1,110 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.


The establishment of this gamete biobank is realized by the Dept Reproduction Biology, and is supported by the supply of sample material of Depts. of Wildlife Diseases and Reproduction Management.

Selected Publications

Zahmel J, Jänsch S, Jewgenow K, Sandgreen DM, Skalborg Simonsen K, Colombo M (2021): Maturation and fertilization of African lion (Panthera leo) oocytes after vitrification. CRYOBIOLOGY 98, 146-151. doi: 10.1016/j.cryobiol.2020.11.011.

Colombo M, Zahmel J, Binder C, Herbel J, Luvoni GC, Jewgenow K (2021): Ovary cold storage and shipment affect oocyte yield and cleavage rate of cat immature vitrified oocytes. CRYOBIOLOGY 98, 181-186. doi: 10.1016/j.cryobiol.2020.11.003.

Colombo M, Zahmel J, Jänsch S, Jewgenow K, Luvoni GC (2020): Inhibition of Apoptotic Pathways Improves DNA Integrity but Not Developmental Competence of Domestic Cat Immature Vitrified Oocytes. FRONT VET SCI 7, 588334. doi: 10.3389/fvets.2020.588334.

Bajerski F, Buerger A, Glasmacher B, Keller ERJ, Mueller K, Mühldorfer K, Nagel M, Ruedel H, Müller T, Schenkel J, Overmann J (2019): Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples. APPL MICROBIOL BIOTECHNOL 104, 131-144. doi:10.1007/s00253-019-10242-1.

Jewgenow K, Braun BC, Zahmel J (2019): Preservation of female genetic resources in feline species. REPROD DOM ANIMAL 54, 13-14.

Fernandez-Gonzalez L, Müller K, Jewgenow K, Zahmel J (2019): Felid-gamete-rescue within EAZA - efforts and results in biobanking felid oocytes and sperm. J ZOO AQUAR RES 7, 15-24.

Müller K, Eder S, Jakop U, Schiller J, Müller P, Bashawat M (2019): Assisted reproduction for felid species conservation – Sperm competences at risk. REPROD DOMEST ANIM. doi:10.1111/rda.13581.

Jewgenow K, Fernandez-Gonzalez L, Jänsch S, Viertel D, Zahmel J (2019): Brilliant cresyl blue staining allows the selection for developmentally competent immature feline oocytes. THERIOGENOLOGY 126, 320-325. doi:10.1016/j.theriogenology.2018.12.021.

Araújo Martins JL, Lopes MD, Ferreira de Souza F, Sossai Possebon F, Wibbelt G, Jewgenow K (2018): Cat preantral follicle survival after prolonged cooled storage followed by vitrification. CRYOBIOLOGY 81, 94-100. doi:10.1016/j.cryobiol.2018.02.004.

Zahmel J, Mundt H, Jewgenow K, Braun BC (2017): Analysis of gene expression in granulosa cells post maturation to evaluate oocyte culture systems in domestic cat. REPROD DOM ANIM 52, Suppl. 2, 65-70. doi: 10.1111/rda.12829.

Luther I, Jakop U, Lueders I, Tordiffe A, Franz C, Schiller J, Kotze A, Müller K (2017): Semen cryopreservation and radical reduction capacity of seminal fluid in captive African lion (Panthera leo). THERIOGENOLOGY 89, 295-304. doi: 10.1016/j.theriogenology.2016.10.024.

Fernandez-Gonzalez L, Jewgenow K (2017): Cryopreservation of feline oocytes by vitrification using commercial kits and slush nitrogen technique. REPROD DOMEST ANIM 52, Suppl 2, 230-234. doi: 10.1111/rda.12837.

Klaus C, Eder S, Franz C, Müller K (2016): Successful cryopreservation of domestic cat (Felis catus) epididymal sperm after slow equilibration to 15 or 10°C. REPROD DOM ANIM 51, 195–203. doi: 10.1111/rda.12666 ISSN 0936–6768.

Sowinska N, Mueller K, Niżański W, Jewgenow K (2017): Mitochondrial distribution, aggregation and activity in oocytes of the domestic cat (Felis catus) after in vitro maturation and vitrification. REPROD DOMEST ANIM 52, 806 - 813. doi: 10.1111/rda.12982.

Lopes CAP, Alves AMCV, Jewgenow K, Báo SN, Figueiredo JR (2016): Cryopreservation of canine ovarian cortex using dimethyl sulfoxide or 1,3-propanediol. THERIOGENOLOGY 86, 1165-74. doi:10.1016/j.theriogenology.2016.04.006.

Fernandez-Gonzalez L, Hribal R, Stagegaard J, Zahmel J, Jewgenow K (2015): Production of lion (Panthera leo) blastocysts after in vitro maturation and ICSI. THERIOGENOLOGY 83, 995-999.

Hribal R, Hachen A, Fernandez L, Zahmel J, Jewgenow K, Braun BC (2014): The influence of recombinant feline oviductin on different aspects of domestic cat (Felis catus) IVF and embryo. THERIOGENOLOGY 82, 742-749. doi:10.1016/j.theriogenology.2014.06.009.

Wiedemann C, Zahmel J, Jewgenow K (2013): Short-term culture of ovarian cortex pieces to assess the cryopreservation outcome in wild felids for genome conservation. BMC VET MED 9, 37. doi:10.1186/1746-6148-9-37.

Wongbandue G, Jewgenow K, Chatdarong K (2013): Effects of thyroxin (T4) and activin A on in vitro growth of cat preantral follicles. THERIOGENOLOGY 79, 824-832. doi:10.1016/j.theriogenology.2012.12.008.

Hribal R, Jewgenow K, Braun BC, Comizzoli P (2013): Influence of culture medium composition on relative mRNA abundances in domestic cat embryos. REPROD DOMEST ANIM 48, 245-251. doi:10.1111/j.1439-0531.2012.02139.x.