Elephants urine bioassays

Abstract A relatively small number of mammalian pheromones has been identified, in contrast to a plethora of known insect pheromones, but two remarkable Asian elephant/insect pheromonal linkages have been elucidated, namely, (Z)-7-dodecen-1-yl acetate and frontalin. In addition, behavioral bioassays have demonstrated the presence of a chemical signal in the urine of female African elephants around the time of ovulation. Our search for possible ovulatory pheromones in the headspace over female African elephant urine has revealed for the first time the presence of a number of known insect pheromones. This search has been facilitated by the use of a powerful new analytical technique, automated solid phase dynamic extraction (SPDE)/GC-MS, as well as by novel macros for enhanced and rapid comparison of multiple mass spectral data files from Agilent ChemStation . This chapter will focus on our methodologies and results, as well as on a comparison of SPDE and the more established techniques of solid phase microextraction (SPME) and stir bar sorptive extraction (SBSE). 

Use of automated headspace SPDE/GC-MS not only enabled the identification in female African elephant urine of a number of known insect pheromones (compounds 2-6, Fig. 2.1), but also revealed the presence of the beetle biochemical precursors to frontalin (2), exo-brevicomin (3) and ent/o-brevicomin (4), thus suggesting a common biosynthetic pathway (Goodwin et al. 2006). Extensive behavioral bioassays must be performed to determine whether any of these compounds is functioning as a pheromone among African elephants. 

URINE BIOASSAYS WITH MALE ASIAN ELEPHANTS... 

The three samples were each presented in discrete areas Im apart on a rinsed concrete slab. These urine bioassays were performed when the ambient temperature was 5-25°C to avoid freezing or rapid evaporation of samples. From the time of sample placement and subsequent release of the test animal, each bioassay was performed for the duration of one hour with a single focal animal. The subject was not present when the samples were placed. In addition, the elephant could not see the observer, thus preventing any bias in behavior that might be caused by the observer s presence. The visible physiological state of the elephant and weather conditions were recorded for each bioassay. Each elephant s behavior was videotaped as he approached the sample areas and while he was within a body length either of the urine samples or the water control san le. 

Fresh urine was collected from Asian elephant females that were at the height of estrus.150 Urine was extracted with an immiscible organic solvent and concentrated. Bioassay involved reconstitution of the extract concentrate in acetone, addition of the acetone solution to (in active) urine samples from females that were not in estrus, and application to concrete slabs or logs. Observers, who were unaware of the sites where samples had been placed, recorded the flehmening responses of test bull elephants. These studies convincingly demonstrated that in Asian elephants the estrous state, and probably the receptivity of the female, is revealed by a substance or substances that can be extracted from the urine 150 of females in estrus. 

Much of our work is focused on the elucidation of the semiochemistry involved in the response of Asian bull elephants to urine of cows in estrus. The number of flehmen responses exhibited by a bull as the result of contact with female urine begins to increase about six weeks prior to ovulation (Hess et al., 1983), probably an indication that the stage of the estrous cycle and the impending receptivity of the female are being revealed to the males via specific chemical components of urine. We have exploited this dependable chemosensory response to develop a unique bioassay to guide chemical fractionation toward isolation and characterization of the responsible substance or substances. The bioassay developed has proved reliable in over 5000 double blind tests using three bulls during a period of four years. 

Bulls were observed in a similar manner during sessions when bioassay test samples were in place and when no test samples were used. In these latter sessions responses to random odor sources (e.g., urine spots from other elephants using the yard) were recorded including the number of re-visitationS to a particular spot. 

The most dramatic exhibition of flehmen by Asian elephant bulls occurs in response to female estrous urine. Bulls exhibit flehmen more to collected estrous urine samples in a bioassay situation than when in the presence of a cow in estrus. A noteworthy exception occurred when a young female experienced her first cycle the number of flehmen responses by the bull was high. An anestrous cow usually elicited brief check(s) a sudden increase in flehmen was observed as she entered the pre-ovulatory stage, rising dramatically just prior to ovulation. 

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