Growing horse embryos to save rhinos
When San Diego Zoo global first announced the Northern White Rhino initiative, I was very excited to be involved in the program. As part of the Reproductive Sciences team, I work mainly with gametes (sperm and eggs or oocytes). So, I wasn’t exactly sure what my part in the rhino rescue project would be. It’s not easy to obtain oocytes from living rhinos, but in the unfortunate event that a rhino passes away, we collect ovarian oocytes.
However, since rhinos usually enjoy very long lives in zoos, these senior females have inactive ovaries resulting in poor quality oocytes. That’s one reason we brought in six young southern white rhinos to the Nikita Kahn Rhino Rescue Center at the San Diego Zoo Safari Park and hired a post-doctoral fellow to help follow their reproductive cycles. She is also training to develop an ovum pick up (OPU) protocol so we can retrieve healthy oocytes from young, living rhinos. Although the oocyte problem was being addressed, there was still one more problem. We have never worked with rhino oocytes or any Perissodactyl (odd-toed ungulate) for that matter. It was time to think outside the box and use a model species for the rhino like we have done with so many other species.
Another member of the Order Perissodactyla, the domestic horse has been extensively studied by a number of our colleagues, who have perfected many of the techniques we need to learn for the rhinoceros.
After some research, we found a few equine embryo laboratories. Two of the top labs in the world are at Colorado State University and at Texas A & M. Both of these labs graciously allowed us to come to their facilities and learn all about horse OPU, oocyte maturation, intracytoplasmic sperm injection (ICSI), and embryo culture. They shared their protocols, and culture medium recipes, and we are very thankful to our collaborators for allowing us to shadow them for a week. By then, we felt a little more comfortable with the idea of working with horse and rhino oocytes.
But we still did not have any hands-on work with horse oocytes. We did some more research and discovered that we have an equine reproduction clinic right in our very own backyard: In Foal Inc. in Hemet. After a visit to In Foal Inc., we asked if perhaps we could buy horse oocytes from them and establish our own equine embryo laboratory at the Beckman Center.
This would be my first opportunity to work with horse oocytes. The one thing I learned was horse oocytes are unlike any other oocytes I have ever worked with, and I have worked with a lot of different species! I remember when I saw a horse oocyte for the first time at Texas A & M; the scientist showed me “a beautiful mature oocyte”, and I couldn’t believe how ugly it was compared to the oocytes I had worked with. I learned right away I needed to adjust my eye (and my brain) to grade horse oocytes differently. I had to learn about cytoplasmic lipid droplet polar aggregation, which makes the oocytes look mottled. I now know it is a good sign in horse oocytes when they have matured in vitro.
Another odd aspect about horse oocytes are that they can only be fertilized by intracytoplasmic sperm injection (ICSI) and not by in vitro fertilization (IVF); the main problem seems to be the inability of sperm to penetrate the egg in vitro. Ongoing research in the field has been aimed at understanding the factors that regulate the process of sperm capacitation, the process that prepares the sperm for egg penetration. But in the meantime, ICSI would be my only option for fertilization. ICSI is commonly used in human fertility clinics and involves the injection of a single sperm into an egg using micromanipulators. I had done this a lot before in other species, so I was fairly confident that I would be able to do it with the horse oocyte.
Starting with a protocol from Texas A & M, we mature the oocytes in the incubator for 30 hours. After maturation, the oocytes are ready to be fertilized by ICSI and cultured in the incubator until reach the blastocyst stage (last stage before uterine implantation). After a few trials (four to be exact) to work out all of the minor problems with equipment, I grew my first horse blastocyst in vitro. Now, I develop multiple blastocysts in vitro weekly. I know it’s just a domestic horse, but it means so much more.
This means that our lab will be ready when we get rhino oocytes. For the rhino we will use the same protocol that is working efficiently with the horse in our lab. My hope is to one day perform ICSI with northern white rhinoceros sperm and eggs to help bring back the population from the brink of extinction. We are running out of time, there are only three left in the world. But we are working hard, dedicating all our time and lab skills to prevent the threat of extinction from becoming a reality.