Captive breeding in giant pandas – Bridging between innovative ART and reproductive biology

We aim to elucidate the secrets of giant panda reproductive biology - particularly the regulation of diapause - employing assisted reproduction techniques (ART) and subsequent in vitro modelling. The acquired knowledge will help to develop embryo transfer protocols in pseudo pregnant females.

The success of the captive giant panda breeding program has been exponentially increasing in the past decades, thanks to the introduction of artificial insemination (AI) and improved oestrus management. The latter includes intensive endocrine monitoring (pinpointing exact moment of fertility) and knowledge on male-female interactions (matching pairs lead to natural mating). A worldwide captive population of approximately 600 individuals was reached end 2019.

Nevertheless, not all pandas are successful breeders, and genetically valuable animals are at risk of being under-represented, hence compromising the population’s resilience. Additionally, surprisingly little is still known about the specific mechanism behind their reproductive peculiarities post oestrus, such as diapause and pseudopregnancy.

In this project we aim to gain knowledge explaining their reproductive biology - particularly the regulation of diapause – through a combined approach of high-resolution ultrasound and state-of-the-art endocrinological techniques, facilitated by assisted reproduction techniques (ART) and subsequent in vitro modelling. The knowledge gained will be used for new techniques of assisted reproduction in the giant panda. The aim is to establish embryo transfer in this highly endangered species. In addition, the IZW is essentially involved in the development of systematic protocols for the diagnosis and treatment of infertility in pandas together with the Chengdu Research Base of Giant Panda Breeding.

The specific project goals are:

• To support the giant panda breeding program at Zoo Berlin through endocrine monitoring (timing AI and pregnancy diagnosis/monitoring), AI and ultrasound training and observations;
• To expand our knowledge on their reproductive biology by enhanced endocrinology with development of diagnostic tools for on-site birth-monitoring;
• To link the endocrine profile to development stages of the embryo, supported by ultrasound observations;
• To develop innovative assisted reproduction technologies (ART) for minimal-invasive recovery of oocytes and to promote early in vitro development of embryos from these oocytes;
• To pursue induction of diapause in vitro and unravel what factors are potentially involved (maternal and foetal);
• And to determine whether the corpus luteum dormancy phase and pseudopregnancy can be exploited as a tool for genetic diversity management.

For this project, the Departments of Reproductive Management and Reproductive Biology have been establishing a successful and sustainable collaboration during the past years. Prof. Hildebrandt is a member of the Academic Committee of the Chengdu Research Base of Giant Panda Breeding and has been elected “Giant Panda Personality of the Year ”, an award at the Giant Panda Global Awards 2019 for his contribution to assisted reproduction of pandas in the Berlin Zoo and in the Chengdu Research Base of Giant Panda Breeding in China. Many project goals have already been met:

• Twin baby pandas were born at Zoo Berlin in August 2019 after successful insemination in April. The pregnancy was confirmed by ultrasound 1 week prior to birth. Intensive hormonal monitoring (PGFM) allowed birth prediction 24 days prior to birth, with another confirmation of timing 30 hours prior to birth.
• In January 2019, the first oocytes were recovered from female giant pandas through trans-abdominal sonography-guided ovum-pick up in Chengdu, China.
• Giant pandas display a period of suspended embryonal development (embryonal diapause) which is flexible in duration, as opposed to European roe deer. The in vitro investigation of the diapause may lead to breakthroughs in unravelling the mechanisms behind arrest and reactivation of the embryo; to this end, the first in vitro study model for diapause in giant pandas is being established. The oocytes collected from pandas will be used to generate embryos and embryonal stem cells. Together with the team of Prof. Hou Rong, Director of the Research Center at the Chengdu Sichuan Key Laboratory of Conservation Biology on Endangered Wildlife in China, the factors relevant for the development of embryos are being investigated. A complementary approach to elucidate the mechanisms of the time-wise determined diapause of the European roe deer is conducted at the field station of the Leibniz-IZW by the Department Reproduction Management and international cooperation partners, using innovative methods. 
• Through the synergistic combination of ultrasound-based (Dept. Reproduction Management) and endocrinological (Dept. Reproductive Biology) techniques we will investigate at which stage of the cycle an in vitro embryo can be transferred into a non-bred pseudo-pregnant female. This will enable us to use genetically overrepresented or less valuable females to carry genetically valuable offspring of individuals that otherwise are unable to produce offspring.

This research is additionally being performed in close collaboration with the Chengdu Research Base of Giant Panda Breeding and Zoo Berlin. Parts of the research (particularly related to endocrine monitoring) are performed based on backgrounds and in collaboration with Ghent University and their respective project partners.