Session 26: Animals
Questions for Pierre Comizzoli
- With the huge media explosion following tank failures a couple of years ago, do you think that the microwave assisted drying would be a viable option for human sperm, eggs and embryos in the near future?
- In humans, there are very strict rules about infectious disease screening when freezing and storing patient specimens. How is that addressed with specimens from multiple exotic species in a frozen zoo setting? ?? ...subsequent question…..Cross species disease too in “surrogacy” yes:)
- Christiani Amorim:Have you ever tried to use your drying system with fragments of ovarian tissue and then assess it by in vitro culture?
What is the concept of precision conservation breeding?
- Pierre: Based on genomics and epigenomics information from each individual from an animal population, the idea is to develop more customized/tailored approaches to optimize the fertility of each individual (and make sure that genes will be transmitted to the next generations). Please read the following article for more information: https://academic.oup.com/biolreprod/article/101/3/514/5322158
- Have any ART animals been reintroduced to the wild?
- Pierre: ARTs have been mainly used to propagate source populations in breeding centers. Some individuals produced by artificial insemination have been then released in the wild.
- How severe is "in breeding" in zoo animals....few living animals must be a problem for diversity?
- Pierre: Inbreeding levels are high in some species kept in captivity because population management and maintenance of genetic diversity are complex. Even if some populations are large enough, only few individuals reproduce, and many do not transmit their genes to the next generation. Also, some breeding programs were started decades ago with very few animals that were related…
- How much can animal ART help our understanding of Human physiology..I hear that hormonal measurements, LH, of PANDAs may signal likelihood of preg loss and miscarriage?..Can Human ART learn from you?
Contact me through ResearchGate for reprints.
- KANTHARAJ M S: Dr Pierre Comizzoli, Do you encourage Xenografting-Hetero, if yes at what level is the risk of cross species diseases and how to overcome it aswell as zoonotic infections.
- Pierre: We do very little xenografting compared to in vitro culture of tissues. We have not studied disease transmissions.
- KANTHARAJ M S: Sir kindly highlight on Glucocorticoid stress responses during the technique used.
- Pierre: Stress is definitely a consequence of any ART procedure in wild animals. We try to minimize it (through animal training when possible) to increase our changes of success.
- Dr Nelson Vincent: In humans, controlled ovarian stimulation is usually done in the female. It is the same done to animals. Is hormonal assay done, especially in the female animals?
I Believe in some studies….Non-invasive assays are done.. from faecal samples
- Pierre: Ovarian stimulation protocols exist but they vary from species to species. We can monitor the natural ovarian cycles and the response to the stimulation by measuring levels of fecal/urine metabolites of steroids.
- Lilia Kuleshova: Happy that you use straw-in-straw it seems that it is low cost efficient straw in straw (Shaw and Kuleshova, 2000). How about oocyte vitrification? Lilia
- Pierre: We vitrify oocytes from different species but we have not been able to produce embryos from those oocytes yet.
- Debbie Montjean: Hi Pierre! Thank you so much for this eye opening presentation. practical question: could you please comment on the technical challenges to overcome in order to allow the utilization of assisted reproduction techniques in animal species..
- Pierre: Salut Debbie! There are many challenges starting with the differences in sizes, anatomy of the reproductive tracts, and physiology of the reproduction. As I explained, we make very slow progress because access to the animals is sometimes limited. Please check my bibliography on that topic on ResearchGate and feel free to ask me reprints.
Questions for Barbara Durrant
- If someone is interested in changing fields from human ART to animal ..how easy is it?
- Hernan Baldassarre: Amazing presentation, thank you so much! In light of the difficulty to collect oocytes from adult rhinos, have you tried sourcing oocytes from prepubertal females when they are small enough to do it by laparoscopy? This is as you know quite successful in farm animal species
- Is the frozen zoo set up with redundant temperature systems?
- Barbara: All tanks have programmable computer displays that we monitor in person or on our computers and phones. Alarms sound if a tank overfills or is underfilled with liquid nitrogen. The alarms are audible throughout the research building and are broadcast to Frozen Zoo staff over their computers and phones. In addition, at night Zoo Security is notified if an alarm sounds. In case of power loss, all tanks are connected to generators to ensure continuous operation. Manual checks of liquid nitrogen levels are conducted routinely to verify computer readouts.
- Are frozen sample banks secure? Are all samples split over different areas of the country / world?
- Barbara: The Frozen Zoo itself is redundant with duplicate tanks in two locations 40 miles apart. Future plans include further distribution of samples for greater security.
- Steven Gellatly: Question for Barbara: Do captive populations of Rhino experience reduced fertility compared with non-captive populations? For instance, are their reproductive life-spans comparable? If yes, how does this influence your artificial breeding programmes
- Barbara: Southern white rhinos born in the wild and brought into human care have excellent reproductive rates. Their female offspring, however, do not. I have included one of our publications on this subject. It implicates a high phytoestrogen diet in zoos as the reason F1 (first generation zoo-born) females do not reproduce well. When we changed our diet fertility was restored in 3 adult rhinos. This dietary influence on fertility is not evident in other rhino species.
Tubbs C, Moley L, Ivy J, Metrione L, LaClaire S, Felton R, Durrant B, Milnes M. Estrogenicity of captive southern white rhinoceros diets and their association with fertility. Gen Comp Endocrinol 238:32-38, 2016.
- diego ezcurra: Do you know Univ.-Prof. Dr. Thomas Bernd Hildebrandt, from
Leibniz Institute for Zoo and Wildlife Research and Dr. Cesare Galli that are already producing embryos from IVM from Northern White Rhinos? We are collaborating by providing a state of the art incubator that not only culture the embryos but also permits a continuous embryo monitoring of NWR development.
- Barbara: Yes, we are very familiar with Thomas and Cesare’s work. Both of them were at the initial meeting in Vienna where the ‘road map’ was developed. What is the incubator you provided Cesare?
- KANTHARAJ M S: Technical limitations in Animal ART compared to Human ART utilization?
- Barbara: The chief technical limitation to animal ART, especially in exotic or endangered species, is the difficulty in obtaining oocytes. In addition, for almost all exotic animal ART, the basics of reproduction are not known or not well described, so each species must be studied first at a very basic level (estrous cycle length, hormone profiles, reproductive anatomy) vs. working with humans that have been extensively studied and documented for decades. Funding is also a limitation - our research subjects don’t pay us to collect and analyze sperm or oocytes/embryos like human patients!
- Brendan Mulligan: Was Oocyte activation used or considered during the ICSI procedure for the White Rhino
- Barbara: We have done ICSI with and without oocyte activation (both electrical and chemical). We currently don’t activate for two reasons: We see that the ICSI and the sperm itself activate the oocyte, and we would have to analyze the embryo to determine if it was a parthenote if we activated the oocyte.
- Natalia Canel: What were the blastocyst developmental rates of ISCNT rhino using horse eggs?
- Barbara: In preliminary experiments we obtained 37% cleavage rates, and 5% went to blastocyst.
- Christiani Amorim: Dr Durrant, did you ever try to xenograft rhino ovarian tissue in immunodeficient mice? Do you think this could be an alternative to obtain oocytes from these animals?
- Barbara: We haven’t tried xenografting yet, but we have frozen ovarian tissue that will be used in those experiments.
- Christina LIM: Hello, once ART in endangered species are successful, how are they going to be introduced in the wild? Will there be challenges there? thank you:)
- Barbara: This is a most important question. Our ultimate goal for the Northern White Rhino Initiative is to produce a self-sustaining herd, first in managed care, then back into native habitat. Before we can return them to the wild, though, we would have to secure the land, the cooperation of the local people, and the protection of the government. This is true of any reintroduced species. We would work with local people and officials, and many collaborators to determine the initial cause of extinction in the area and put into place a concrete, funded plan to prevent future extinction.
- Mary Zelinski: For Dr. Durrant: are you considering in vitro follicle culture/maturation as an option for the Natural Gamete side of your Roadmap for preserving rhinos?
- Barbara: Hi Mary! This is an interesting idea that we have not considered. It could be quite useful for intact ovaries from post-mortem collections. Dissection of whole follicles during in vivo collection of oocytes is not possible with the current instrumentation. We would be very interested in your ideas for follicle culture.
- Dee Kini: From your presentation I think animal fertility preservation is far more advanced than in humans. Do you agree?
- Barbara: I wish that fertility preservation was as advanced in animals as it is for humans. It may be close for some domestic or laboratory animals, but unfortunately, it is not for endangered species. There are so many species to study and so few labs working in this area that our progress is necessarily slower than in humans. The best we can do now is to freeze sperm, oocytes and reproductive tissue for the future when techniques have been perfected for related species (for example, in the horse as a model for the rhino).
- Panagiota Efstathiou: Thank you so much for these amazing presentations! Could we please have more talks on assisted reproductive technologies for endangered animal conservation in the future?
- Barbara: I have made a couple of suggestions for future speakers to the organizers of the i3 webinars.
- Pauline Kibui: How long does it take to mature rhinos oocytes?
- Barbara: We see maturation between 30 and 36 hours of in vitro culture.
- Jarrod Mckenna: How do the new generation of conservations, such as myself (PhD candidate) get into the field of ART-based conservation? Especially in a funding-restricted COVID climate. Should we look to build skills in cattle/domestic species first?....this would be great for both during the general discussion?
- If someone is interested in changing fields from human ART to animal ..how easy is it?
- How can WE work more closely #OneScience
- Do you have collaborations with human IVF labs? What can embryologists in the human field do if they are interested in helping out or volunteering?
- Kay Spies: How do you choose the wildlife species of interest to examine and research reproductive physiology?
- From the I3 team: If we have another session..what would YOU like us to concentrate on?
- Barbara: I would be very interested in sessions on mtDNA quantification, transfers, deactivation, etc, that would be applicable to interspecies SCNT.
- Pierre: Barbara also mentioned microbiomes. I would be happy to join a discussion on that topic. In addition, the integration of genomic and epigenomic tools into animal conservation is a very hot topic.
- Who owns the tissue samples? Are there disputes?
- Barbara: The ownership of tissue samples is determined by the ‘owners’ of the donor animals. If samples are sent to us from another zoo we ask for them to be donated for research purposes. If they prefer to retain ownership we process and store the tissue until they take ownership. It becomes a bit complicated if offspring are produced, but curators at each facility decide who will own each offspring.
- The marine mammals must be in a bottleneck with few natural captured animals to replenish the genetic stock..how established is an AI system for this?
- Barbara: AI has been successful in only a few marine mammal species (dolphins and killer whales). Due to animal welfare concerns, AI has been discontinued at SeaWorld facilities. Overall, AI is not considered a feasible method to replenish genetic stock in captured animals.
- What's this "Laser process" of warming vit eggs?
- Barbara: his system of rapid warming vitrified oocytes and embryos involves subjecting them a thaw rate of 107°C/minute thaw with a laser pulse.
- Pierre: Gold nanoparticles have to be added to the extenders in order to conduct the LASER heat as fast as possible. The warming technique works very well for large eggs or embryos (from fish for instance). We are exploring the same technique for ovarian and testicular tissues.
- Jc: What are the bottlenecks for further (and preferably rapid) R&D? Is it just financial support?
- Barbara: Funding is certainly a major bottleneck for developing assisted reproduction in endangered species. Adequate funding would allow more labs to conduct much-needed basic research. Working with endangered species means there are far fewer individual animals to study (compared to mice, cattle, humans, etc), which slows progress. The welfare of each individual of an endangered species is of utmost importance, which limits the number and kind of procedures that can be done.
- Jc: Are there graduate and post-graduate training programs for conservation studies?
- Pierre: There is a Smithsonian-Cornell Joint Graduate Training Program on that topic. Feel free to contact me for more information.
- Jc: Over 400 small mammal species are currently threatened and many may become extinct soon. Why is it that the focus is mostly on large mammals? This problem seems to be also in conservation efforts of marine life. Large animals seem to have priority! Is this because the conservation efforts are usually zoo or aquarium/dolphinarium - centered?
- Barbara: This is a great question. I hope Pierre will also respond. While overall you are correct, in San Diego we have a number of projects with small (even tiny) species of mammals, for example the Pacific pocket mouse, the Hawaiian Akikiki bird, the diminutive Mountain Yellow-legged frog and others. Perhaps because the larger animals are more familiar to the public they get more press.
- Pierre: Some programs are driven by donors or foundations who have an interest in some species. It usually is on mega vertebrates. However, there are many programs for small mammals, amphibians, reptiles, etc. there are less publicized and do not catch people’s attention.
- Jc: There are currently over 16,000 threatened (nearly extinct) registered species (plants and animals) not including a vast number of invertebrate, insect, plant and microbial organisms. It seems that when an animal is considered ‘cute’ (Koala, Rhino, Panda) or is of value to the economy (Bee), conservation efforts find (some) public and private support. How can we educate each other better?
- Barbara: Education about the diversity of plant and animal species is a continuing challenge. More popular press articles about the fascinating aspects of little-known species is a good place to start. Emphasis on plants is vitally important, but often overlooked. Simply pointing out that all animals depend on plants, whether they eat them, nest in them, or eat vegetarian species can start that conversation.
- Pierre: Even if the conservation efforts are focused on a single species, the other species sharing the same natural habitat will benefit from the conservation actions. For instance, while we save the giant panda in the bamboo forest, we also save many other species.
- diego ezcurra: did you hear from the In Vitro Maturation media developed in the university of Missouri by a team of researchers including my friend Dr. Clifton Murphy? They claimed a huge increase in the maturation rate
- Barbara: what species are they working with in Missouri?
- Janice Vilela: For both Pierre and Barbara: do you see this effort of increasing populations of now endangered species one day leaving the zoo environment, and these animals being able to live in the wild? What are your thoughts on that?
- Pierre: Yes, this is the ultimate goal. However, it will always be important to keep safety populations (live animals and Genome Resource Banks) of the reintroduced species. This is the best way to prevent future issues in the wild. The reintroduction program of the black-footed ferret is following that model.
- Natalia Verónica Rubio: As a biologist who works in an IVF clinic as an embryologist, living abroad, how could I get involved with conservation or collaborate in any way?
- Barbara: You can research available programs in your area - a zoo, a university department specializing in conservation or ecology, field biologists, rescue organizations - these are all organizations that could use your skills and interests.
- Steven Gellatly: Are sperm counts declining in these 'exotic' animals?
- Barbara: I am not familiar with data indicating declining sperm counts, perhaps Pierre will have more information.
- Pierre: Yes, sperm counts are decreasing in some zoo populations, but we know why. It is due to the increased level of inbreeding. We also have noticed the same trend in reintroduced black-footed ferrets, again, because of the inbreeding levels.
- Philip Damiani: Do you consider Inducible pluripotent stem cells with the use of somatic cell lines given some of the success with other species?
- Barbara: Hi Phil! I’m not sure what you are asking. The iPSCs we work with originate as skin cells cultured to fibroblasts. Feel free to contact me at the zoo.