Putting eggs in a basket, step 2: Make the eggs happy – oocyte maturation and fertilization in vitro

For over 3 years now I have been preparing for the day when I receive fresh rhino oocytes. Sure, I’ve acquired oocytes from post mortem rhinos. But fortunately our rhinos live a long and happy life and by the time I get their oocytes, they are about 50 years old. It’s comparable to a woman trying to have a baby at 70 years old. Very unlikely. 

When I heard that we were going to bring Dr. Morné de la Rey from EmbryoPlus in South Africa (Building an international team to save the Northern White Rhino (NWR)) to our facility to collect oocytes from our young rhinos via ovum pick-up (OPU), I was ecstatic. Briefly, during OPU a needle is passed into a follicle on the ovary (follicles are structures that hold the oocytes) and the follicle contents are aspirated (Putting eggs in a basket, step 1: Recover the eggs – Ovum Pickup (OPU) procedure). All these years spent practicing with horse oocytes was finally going to pay off. 

My lab group learned all about horse in vitro maturation (IVM), intracytoplasmic sperm injection (ICSI) and in vitro culture (IVC) from our collogues at Colorado State University, Texas A & M, and In Foal Inc. The horse is the closest domestic perissodactyl related to the rhino. It only seemed logical to start there. We visited each group, brought back all the information we learned and applied it in our lab. Within a month of practicing on my own I was able to produce horse blastocysts (the final stage of embryo development before implantation). I of course was thrilled, but I knew it was only the beginning of my journey to produce northern white rhino embryos. With the horse model well established in the lab, next would be the southern white rhino. And the time had finally come. 

Dr. de la Rey aspirated oocytes from four female southern white rhinos. The first was Dakari, who ‘donated’ 2 oocytes. Four oocytes were collected from the second rhino, Utamu, and 3 were recovered from the third rhino, Nikita. The last rhino, Livia, gave us an astounding 13 oocytes! This was Dr. de la Rey’s personal record and perhaps a world record. This gave us a total of 22 oocytes to work with in the lab. Never in my wildest dreams did I ever think I would have so many fresh rhino oocytes. The oocytes were divided into two different maturation media to test different protocols. The maturation media contain hormones similar to those the female would produce as the oocytes mature and become ready for fertilization. 

After 36 hours of in vitro maturation culture I removed the cells surrounding the oocytes so I could visually confirm maturation by the presence of a polar body. It was then that I realized I had a clear winner between the two maturation media I tested. One resulted in a 50% maturation rate, whereas only 9% of the oocytes cultured in the other medium matured. This was an important step knowing which medium to use in the future. 

Now the oocytes were ready to be fertilized. I injected all the oocytes with a single sperm using micromanipulators (I am an animal embryologist). This process is commonly used in human fertility clinics, and is known to be the best way to fertilize horse oocytes, so we decided we should use this method with the rhino oocytes as well. After fertilization the rhino oocytes were placed into culture medium in the incubator so that the embryos begin to divide. The culture medium contains sugars, amino acids and antibiotics similar to the fluid in the uterus that allow the embryos to divide and develop to the blastocyst (In vitro-what does it mean?).   

After a few days in the incubator, I checked on the oocytes. Unfortunately, only one oocyte cleaved, but at the same time ONE OOCYTE CLEAVED!!! This was a huge success for us. Not only did we determine the best maturation medium to use with rhino oocytes, but we also achieved cleavage. I know next time we get fresh rhino oocytes we will have a better idea what to do and each time we will have better success. It only takes some time and, of course, practice.  One day soon I will produce a southern white rhino blastocyst and perhaps in the future a northern white rhino blastocyst. 

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