Flavor Chemistry of Animal Foods

4 Isolation, Identification, and Biological Activity Assay of

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Chemical Fractions from Estrus Urine Attractive to the Coyote E. L. MURPHY, ROBERT A. FLATH, DALE R. BLACK, THOMAS R. MON, and ROY TERANISHI Western Regional Research Laboratory, Agricultural Research Service, U. S. Department of Agriculture, Berkeley, CA 94710 R. M. TIMM and W. E. HOWARD University of California, Davis, CA 95616 Most of the sheep killed by predation in the United States are by coyotes. Ranchers or farmers who have recently quit the business of raising sheep most often blame the lack of control of coyotes as being the precipitating cause for their decision. The greatest problem of using a device or chemical to affect coyote populations is to get the attention of the coyote. Excessive numbers of chemical baits must be dropped or a great number of "Coyote Getters" must be set to reach one coyote. Treating with an effective attractant would significantly reduce the cost and work involved. Further, it is very desirable that the attractant be specific for coyotes to reduce the injury and death to non -target species such as skunks, bobcats, eagles and pet dogs. Many attempts have been made to concoct brews or mixtures to be irresistably attractive for the coyote as he passes a dropped bait or trap. Mixtures of blood, animal organs and urine brewed or fermented in numerous ways have been used for years by ranchers and trappers with varying success. One ingredient common to many scent bait mixtures has been coyote urine. Several researchers have noted that significantly more coyotes have been caught with estrus than with non-estrus urine. This paper reports the initial efforts to isolate and identify an attractant from estrus urine which might prove specific for the coyote. Experimental Estrus Urine Collection. Female coyotes were confined singly in small cages with wire bottoms below which was fitted a stainless collection tray which sloped to empty into a one pint glass canning jar. In order to reduce fecal and drinking water contamination of the collected urine, the coyotes were fed in the morning and in the afternoon all food scraps and water were removed and the cages and collection trays cleaned. The jars were then put in place and urine collected overnight. In the morning the jars of urine were collected, frozen and stored at -20°C. The coyotes were then fed and the collection cycle repeated. For the purposes © 0-8412-0404-7/78/47-067-066$05.00/0 Bullard; Flavor Chemistry of Animal Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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of t h i s study u r i n e was considered estrus urine when c o l l e c t e d i n a period between the appearance and disappearance of vaginal bleeding. Chemical F r a c t i o n a t i o n . The frozen urine was thawed and pooled in one 50 £ batch f o r chemical f r a c t i o n a t i o n . It was made basic (pH 12) with potassium hydroxide and the basic and neutral compounds extracted with d i e t h y l ether according to the e x t r a c t i o n scheme o u t l i n e d i n Figure 1. The gel f r a c t i o n separated from the coyote urine a f t e r i t had been made b a s i c and was recov ered by f i l t r a t i o n of the basic u r i n e . The b a s i c compounds were separated by e x t r a c t i o n with 6N h y d r o c h l o r i c a c i d leaving the neu t r a l compounds i n the ether r e s i d u e . The o r i g i n a l ether e x t r a c ted b a s i c u r i n e was then made a c i d (pH 2) and the a c i d i c com pounds extracted with d i e t h y l ether. The a c i d i c , basic and neu t r a l f r a c t i o n s were s t r i p p e d of ether solvent by d i s t i l l a t i o n . Bioassay of Urine F r a c t i o n s . The degree of a t t r a c t i o n of the estrus u r i n e f r a c t i o n s , a c i d i c , b a s i c , neutral and urine r e s i due, was estimated by presenting each f r a c t i o n with i t s s o l v e n t c o n t r o l in a randomized sequence to each of s i x coyotes i n r e p l i cated t r i a l s . Each coyote was r e l e a s e d f o r t h i r t y minutes i n t o a large pen, 6m χ 20m, Figure 2, containing the t e s t urine f r a c t i o n with i t s solvent control i n separate portable scent s t a t i o n s on the ground and i t s behavior recorded by a hidden observer as i t approached each scent s t a t i o n ( l j . Time at each scent s t a t i o n as well as behavior such as r u b - r o l l i n g , chewing, s n i f f i n g , s c r a t c h i n g and scent marking was recorded by the observer. Three male and three female coyotes were used. Component I d e n t i f i c a t i o n . In preparation f o r gas chromato graphy the a c i d i c urine f r a c t i o n was reacted with diazomethane produced from DIAZALD, N-methyl-N-nitroso-p-toluenesulfonamide, A l d r i c h Chemical Company (2). As the diazomethane was generated by the a d d i t i o n of potassium hydroxide, the ethereal diazomethane s o l u t i o n was d i s t i l l e d i n t o an i c e bath-cooled r e a c t i o n f l a s k which contained the urine acids f r a c t i o n . The production of diazomethane was continued u n t i l a b r i g h t yellow c o l o r p e r s i s t e d i n the r e a c t i o n f l a s k . The r e a c t i o n mixture was allowed to s i t i n a hood overnight during which time the i c e melted allow ing the r e a c t i o n mixture to come to room temperature ( 2 3 ° C ) . The ether solvent was then removed by d i s t i l l a t i o n on a steam bath. P r e l i m i n a r y gas chromatographic runs were made on a Hewlett/Packard, Model 5831 A, gas chromatograph. Separation of the methyl e s t e r i f i e d components i n the a c i d i c u r i n e f r a c t i o n was made on a 0.03 i n . i . d . X500 f t s t a i n l e s s s t e e l open tubular c o l umn coated with methyl s i l i c o n e o i l , SF 96-50 (General E l e c t r i c ) containing 5% Igepal CO-880 (General A n i l i n e and Film) using a flame i o n i z a t i o n d e t e c t o r . A f t e r sample i n j e c t i o n , the columns were held at 76°C f o r 30 min; then the oven tenperature was

Bullard; Flavor Chemistry of Animal Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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COYOTE URINE

1. BASE 2 ETHER INITIAL ETHER EXTRACT

BASIC EXTRACT


ACID

ACIDIC EXTRACT

ETHER EXTRACT

(NEUTRALS)

ETHER EXTRACT

(BASES)

ETHER EXTRACT

(ACIDS)

URINE RESIDUE

(RESIDUE)

AQUEOUS EXTRACT

Figure 1. Urine fractionation procedure

Figure 2. Coyote test area


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programmed at 2°/min to 210°C. T e n t a t i v e i d e n t i f i c a t i o n s were made from mass s p e c t r a l data obtained by coupling the e f f l u e n t end of the gas chromatograph column to a quadrupole-type mass spectrometer through a s i n g l e stage s i l i c o n e membrane i n t e r f a c e (Quad 300, E l e c t r o n i c Associates Inc.) as described by F l a t h , Forrey and Guadagni (3), and F l a t h and Forrey (4).


Results and Discussion An e r r o r which i s d i f f i c u l t to avoid i n the c o l l e c t i o n method described i s the i n t r o d u c t i o n of a r t i f a c t chemical compounds i n t o the urine during c o l l e c t i o n which show up in the l a t e r chemical analysis. Contamination of the c o l l e c t e d urine by h a i r , f e c e s , d r i n k i n g water, food s c r a p s , metabolic byproducts of m i c r o b i a l fermentation and a i r o x i d a t i o n can be reduced by design of the c o l l e c t i o n procedure. To reduce chemical and m i c r o b i a l contaminat i o n f u r t h e r , u r i n e can be c o l l e c t e d under a t h i n l a y e r of toluene in a r e s e r v o i r with continuous urine flow i n t o a r e f r i g e r a t e d c o l l e c t i o n j a r where i t i s r a p i d l y f r o z e n . Experience with human urine c o l l e c t i o n i n d i c a t e d l i t t l e change in chemical composition of frozen urine a f t e r several months storage. When whole undiluted coyote urine i s gas chromatographed on high r e s o l u t i o n c a p i l l a r y columns with s o p h i s t i c a t e d computer comparison of the recorder t r a c i n g s , no s i g n i f i c a n t d i f f e r e n c e s can be demonstrated between e s t r u s and non-estrus u r i n e . Theref o r e , there must be an extensive reduction in the number of chemi c a l compounds by p r e f r a c t i o n a t i o n before i d e n t i f i c a t i o n of the i n d i v i d u a l chemical components can be attempted. One of the o l d e s t and most common approaches f o r the i s o l a t i o n and i d e n t i f i c a t i o n of the chemical cause of an a c t i v i t y i s the step by step chemical or physical separation of a natural products mixture while f o l l o w i n g the a c t i v i t y by an e f f e c t i v e bioassay. At each step the chemist must wait before proceeding with the next separat i o n f o r the r e s u l t s of the bioassay (often conducted by other members of a m u l t i - d i s c i p l i n e s c i e n t i f i c team) to i d e n t i f y the chemical f r a c t i o n which contains the major p o r t i o n of the a c t i v i t y . The chemical s e p a r a t i o n , Figure 1, produced four coyote urine f r a c t i o n s f o r t e s t i n g : a c i d s , bases, n e u t r a l s and urine r e s i d u e . The design and operation of a s u i t a b l e assay f o r the measurement of b i o l o g i c a l a c t i v i t y almost always poses the greater problem f o r the chemist r a t h e r than the various chemical separations. Because i t i s a part of his t r a i n i n g and he has the necessary s p e c i a l i z e d equipment, the chemist should p a r t i c i p a t e in the prepa r a t i o n of the t e s t samples. He can advise as to choice of s o l vents, exact c o n c e n t r a t i o n s , v o l a t i l i t y in transport and p l a c e ment, s t a b i l i t y to heat and oxygen of the a i r as well as in poss i b l e sources of contamination. But at the t e s t pen, using t e s t animals as v a r i a b l e and complex as the coyote, the chemist gives way to the s p e c i a l i s t in w i l d l i f e biology as to animal care and



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handling, t e s t design, accurate and o b j e c t i v e observation and most important, the i n t e r p r e t a t i o n of behavior. Figures 3 and 4 (_5) i l l u s t r a t e that of the four t e s t f r a c t i o n s , the acids f r a c t i o n e l i c i t e d the most time spent by the coyotes i n s n i f f i n g or r u b - r o l l i n g behavior. With c o n s i d e r a t i o n as to both t o t a l behavioral events, Figure 5, and time spent at the t e s t odor, Figure 6, a l l coyotes were a t t r a c t e d more by the acids f r a c tion. Comparison of r u b - r o l l i n g behavior of the acids f r a c t i o n with the urine r e s i d u e , which i s almost background, i n d i c a t e s that the e x t r a c t i o n procedure removed most of the compounds r e s p o n s i b l e f o r t h i s behavior. It i s i n t e r e s t i n g that the acids f r a c t i o n exh i b i t s greater r u b - r o l l i n g s t i m u l a t i o n than the commercially a v a i l a b l e coyote trap scent. Both bases and neutrals f r a c t i o n s were lower in s n i f f i n g or r u b - r o l l i n g response. Scent marking (Figure 7) and scraping (Figure 8) behavior was demonstrated by the coyotes longer in response to the bases f r a c t i o n (.5). This i s c l e a r l y a d i f f e r e n t behavior to another f r a c t i o n of the urine and i s d i f f i c u l t to e x p l a i n , other than that t h i s f r a c t i o n may contain one or more chemicals a s s o c i a t e d with t e r r i t o r i a l marking. In a l l cases, the coyotes paid very l i t t l e more a t t e n t i o n to the gel f r a c t i o n than to the ether c o n t r o l or urine r e s i d u e . Pen t e s t s have several disadvantages f o r t e s t i n g u r i n e . The close r e s t r a i n t of even a large observation pen and the necessary occasional movement of personnel undoubtedly produce d i f f e r e n c e s in the behavior of penned coyotes as compared to the animal i n i t s natural h a b i t a t . F u r t h e r , pens tend to become u r i n e saturated from scent marking from previous t e s t i n g such that the urine f r a c t i o n s are being t e s t e d against a high background "noise l e v e l " . Again t e s t design may be used to compensate f o r some of the d i f f i culties. The t e s t coyotes should be housed and tested i n a remote area f r e e from excessive v e h i c l e and human t r a f f i c . Some coyotes l i k e some people are anosmic; t h e r e f o r e , only animals should be chosen f o r t e s t i n g which r e a d i l y respond to some standard odor of proven a t t r a c t a n c y and f a i l to demonstrate s i g n i f i c a n t i n t e r e s t in a blank t e s t sample of low odor content. Test s t a t i o n s or l o c a t i o n s of t e s t samples should be moved about i n the pen. T e s t samples should be presented i n a randomized sequence and r e p l i cated with as many animals as permitted by the resources and t e s t o b j e c t i v e s of the experiment. The chromatogram by g a s - l i q u i d chromatography, Figure 9, i n d i c a t e d over t h i r t y major peaks or chemical compounds i n the acids f r a c t i o n of the estrus coyote u r i n e . Mass s p e c t r a l data permitted t e n t a t i v e i d e n t i f i c a t i o n of the methyl e s t e r s of a s e r i e s of short chain f a t t y a c i d s , CO-C-.Q, together with aromatic compounds as present. Table 1 l i s t s 19 t e n t a t i v e i d e n t i f i c a t i o n s of compounds i n the acids f r a c t i o n . Because other i n v e s t i g a t o r s {6) have reported that an a r t i f i c i a l mixture of s i m i l a r f a t t y acids demonstrated s i g n i f i c a n t a t t r a c t i o n of coyotes i t w i l l be i n t e r e s t i n g to prepare a mixture of the f a t t y acids i d e n t i f i e d i n coyote u r i n e 1n the exact r a t i o that they are 1n


MURPHY

E TAL.

Chemical

Fractions

from

Estrus

Urine

6 C O Y O T E S IN 48 INDIVIDUAL TESTS

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Flavor Chemistry of Animal Foods

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