That’s the big question: did female cheetah Lindiwe successfully breed, and is she currently pregnant? Just last month we saw strong interest between Lindiwe and one of our proven breeder males, Noka. Lindiwe is a young cheetah, and this would make her a first-time mom, which is very exciting! From our point of view, the most important thing about getting a naïve female cheetah to breed is that once she breeds, she tends to continue having litters throughout her reproductive years. So the question remains: was Lindiwe actually in estrus and did not allow breeding, or did she simply not come into estrus during our breeding attempts? To answer the question, follow me into the Behavioral Biology Endocrinology Lab.
This lab offers us an opportunity to test hormone levels in a variety of exotic species. Hormone research adds an additional dimension to our research projects by providing another tool for unraveling the mysteries of animal behavior. When we are trying to test reproductive hormone levels in cheetahs, our preferred method involves non-invasive hormone sampling, a technique where the animals are unaware that we are testing their hormones. Blood collection can be a stressful procedure, which often results in adverse affects on reproductive hormones and/or behavior, so we usually sample urine, feces, and sometimes saliva and hair instead. The cheetahs go about their normal daily routine having no idea that we are testing their hormones levels!
Fecal (or poop) samples are collected by our cheetah care staff, promptly frozen, and brought to the lab for hormone testing. The first thing I do is dry the samples on a lyophilizer, a really big, fancy freeze drier that removes all the water from the fecal samples. I then crush and sift the dried fecal samples before weighing out a specific amount. I now have dried, weighed fecal material in a test tube and am ready to extract the hormones held within. There are many ways to do this, and they usually involve using a solvent in combination with some type of force. In our cheetah samples, I add solvent to the fecal material and mix (vortex) or heat the samples. At the end of this extraction process I am left with a test tube full of solvent that contains not only extracted hormones but also other extracted compounds. The trick is for me to find an appropriate laboratory procedure (or assay) that I can use to examine the concentrations of the specific hormones in question (in this case, female reproductive hormones). In humans, hormone assays are generally routine, but in exotic animals, extractions and assays can vary both between species and within species, depending on the hormones of interest and what biological sample they are from (fecal, urine, or saliva).
So back to the original question: was Lindiwe physiologically in estrus during the time she was showing the appropriate female sex behaviors? To answer this question, I needed to test her fecal samples for the hormone estradiol (a specific form of estrogen). In most species I could usually determine if the animal in question is cycling by looking at progesterone levels. But cheetahs are more complex as they are induced ovulators (see Cheetah Breeding Excitement). As such, their progesterone levels remain low unless a follicle, or egg, has actually been released from the ovary. To complicate matters further, when we study hormone levels in urine or feces, we usually only see metabolized hormones because we are looking at a waste product of the body. These are different from the hormones moving around in the bloodstream (known as parent or non-metabolized hormones) that are much easier to measure. Examining the concentrations of estradiol metabolites can be complicated and tricky because different animal species often metabolize hormones uniquely. These are the types of challenges we face in our endocrinology lab when studying hormones in exotic species.
After testing Lindiwe’s samples for hormones to see if she was truly in estrus during our breeding attempts, it appears that her rolling and tail flicking behaviors were somewhat misleading. Her hormone levels were quite low on the days she showed such estrus behavior. Interestingly, her hormone levels did go up, indicating a mild, short estrus, after we had stopped our breeding attempts. Welcome to the frustrating world of cheetah breeding!
We see a wide range of estrus behaviors varying from “silent” to “clear,” and some of our continual challenges include trying to decode the differences in behaviors between the females in our collection. We had very high hopes that Lindiwe was truly in estrus and would allow breeding by Noka, but alas, they did not end up breeding. We will continue our breeding attempts between the two, so please cross your fingers and hope for the best.
Corinne Pisacane is a senior research technician in the Behavioral Biology Division of the San Diego Zoo Institute for Conservation Research.