Physiology & Behawor, Vo! 45. pp 1207-1214 Maxwell Pergamon Macmillanplc.

1989 Pnnted an the U S A

0031-9384/89 $3 00 + 00

The Role of the Flehmen Response in the Behavioral Repertoire of the Stallion
C A T H I C. S T A H L B A U M AND KATHERINE A. HOUPT 1

New York State College o f Veterinary Medicine, Cornell University, Ithaca, N Y 14853
R e c e i v e d 28 N o v e m b e r 1988

STAHLBAUM, C C. AND K. A. HOUPT The role of the Flehmen response m the behavioral repertoire of the stallion PHYSIOL BEHAV 45(6) 1207-1214, 1989 --The role of the Flehmen response an equine behavior was investigated under field and laboratory condmons. In Experiment 1, a field study made of five stallions on pasture with between three and eighteen mares each during the season indicated the following 1) The Flehmen response was most frequently preceded by nasal, rather than oral, mvestlgataon of substances; 2) The stallions' rate of Flehmen vaned with the estrous cycles of the mares; 3) The rate of Flehmen response did not show a vanataon with rime of day, and 4) The Flehmen response was most frequently followed by marking behaviors rather than courtship behaviors. The results suggest that the Flehmen response is not an lmmethate component of sexual behavior, e.g., courtship of the stalhon but may be involved in the overall momtonng of the mare's estrous cycle Therefore the Flehmen response may contribute to the chemosensory pnmmg of the stalhon for reproduction In Expenment 2 stalhons were presented with unne or feces of mares in various stages of the reproductive cycle as well as with their own or other males' urine or feces The occurrence of sniffing and Flehmen was used to deterlmne the discriminatory ability of the stalhons. Stalhons can differentiate the sex of a horse on the basis of its feces alone, but cannot differentiate on the basis of unne This abihty may explain the function of fecal marlong behawor of stallions Flehmen Sex Horse Unne Olfactlon Stallion

THERE has been a considerable increase in our knowledge of the function of the Flehmen or lipcurl response since Estes (4) first hypothesized its role. Although Flehmen occurs in all ungulates (with the exception of pigs that exhibit a gape response in similar situations), almost all of the recent studies have been on ruminants: goats, sheep, deer and cattle. Generally these bovids live in separate sex groups; the males interacting with the females only during the breeding season, a fact that may necessitate olfactory recognition of estrus. Horses live in harem groups where visual cues and familiarity with mares may be more important to estrus detection. Flehmen was first described in the horse (15) and its occurrence has been noted in feral horses (5,19). It has been shown that Flehmen is exhibited mostly by stallions and its frequency is diminished by gelding (18). Recently Mannier et al. (12) have shown that mares are intermediate in their response to equine elimination; they Flehmen and sniff less than stallions, but more than geldings. The association of Flehmen with the vomeronasal organ secretions and the detailed anatomy of that organ have been described by Lindsay and Burton (11). The microscopic anatomy of the equine vomeronasal organ has also been described (7,17). In the horse the incisive duct opens only into the nasal cavity rather than into both the oral and nasal cavities as in most ruminants. Whether these social and anatomical differences are reflected in the circumstances in which Flehmen occurs in horses has not been investigated.

We hypothesize that Flehmen might occur in different circumstances and have a different function in horses than in ruminants and that the function would reflect the social grouping and the proximity of mares. To test the hypothesis, the behavior of adult stallions towards mares and their excretions should be investigated; therefore, a field study was conducted to determine what substances or circumstances stimulated Flehmen and the relation of Flehmen rate to the sexual receptivity of the mares m each stallion's harem. The results of the field study led us to perform behavioral bloassays in confined stallions to further test which types of excretions elicit the response most frequently. Of particular interest was the question of whether or not the rate of Flehmen to urine or feces varied with the sex and reproductive state of the donor. More specifically, the association between the frequency of Flehmen by the stallion and the mare's estrous cycle was assessed in the field study, whereas the bioassay sought to determine the ability of the stallion to discriminate the sex and reproductive state of the urine or fecal donor. METHOD

Experiment 1: The Occurrence of Flehmen in a Seminaturalistic Situation

Focal observations were made on five sexually experienced pony stallions (A-E) with mares and their foals on pasture (1 to 9 hectare) from late May through mid-August. All but three of the

IRequests for repnnts should be addressed to K. A. Houpt, Department of Physiology, NYSCVM Cornell University, Ithaca, NY 14853-6401.

1207

1208

STAHLBAUM AND HOUPT

TABLE I
THE RELATION OF ~ E H M E N RATE/DAY TO DAY OF COPULATION

Stallion
B

- 5 24 31 28035

- 4 50 34 42080

- 3 34 35 35005

- 2 20 34 27~070

Day of Copulation - 1 0 40 45 43~025 38 69 54~16

2 38 34 36~020

3 28 32 30020

4 32 59 4613

5 34 32 33001

A Mean

42 31 37078

The number of Flehmen responses recorded dunng 2 hr of observations/day The date of copulation with each mare was considered day 0 for that mare. and might be day - 4 for another mare and + 2 for still another The mean for all days [ - 5 to +5) as calculated for each stalhon

mares were reproductively cycling during part of this period, the other three were pregnant throughout the study The number of cychng mares varied as 1) some mares foaled and came into estrus, 2) mares became pregnant, and 3) mares were added to the pasture The stalhons and the numbers of mares in their respective pastures were" Stallion A - - 1 3 mares, Stallion B--imtlally 13 and later 18 mares; Stalhon C - - 4 mares; Stallion D - - 3 or 4 mares as one was removed and another added; stallion E - - 2 mares. Four daily half hour observations of stallions A and B were conducted during the hours 0400-0600, 1130-1330, 1930-2130 and 2330-0130 from late May through mid-August. Daily 15 minute observations of stallions C, D, and E were made between 0500 and 0700 from June 1 through June 16. From June 16 to June 20 these observations were extended to 30 minutes. Beginning on June 21 a second observation period was added between the hours of 1930-2130 Observations commenced when the stalhon was first sighted In all, 330 hours of observation of stalhons A - E were conducted. All occurrences, and durations where appropriate, of the following behavior patterns were recorded Nasal mvestigato~ behavtor. Nasal investigatory behavior patterns included both sniffing and Flehmen responses. A sniff was recorded if the stallions's nostrils were within 15 cm of an object while he performed rapid inhalations. Sniffs were classified into one of the following categories "to urine," recorded if the sniff occurred at an area where a horse was observed to urinate or where a pool of urine was visible, " t o feces," recorded only ff feces could be detected where the stallion sniffed; " t o ground," recorded in areas where neither feces or urine were obvious, "to a mare's ano-genital region," "nasal-nasal sniffs," or "to another part of the female." Any other sniffs were classified as a "sniff to other "' The Flehmen response was characterized by the curhng back of the upper lip accompanied by deep inhalations and exhalations Flehmen responses were divided into categories depending on the behavior that preceded the response. These categories included sniffing urine, sniffing feces, sniffing a mare's ano-gemtal region, sniffing a female elsewhere, sniffing the ground, or behavior other than a sniff. Sexual behawor. The occurrence, but not duration, of all penile letdowns, erections, mounts, and xntromisslons were recorded Penile letdowns were classified as occurring within 60 seconds after a sniff, Flehmen response or other behavior Markmg behavior. All occurrences of eliminations by the stallion were recorded These eliminations were considered to be marking behaviors only if the behavior immediately preceding the elimination was a sniff or a flehmen response, or if the stallion stood with hind limbs extended over urine or feces, and deposited a small quantity of urine m a short burst or defecated on the excrement. Marking behaviors consisted of unne marking on urine, feces, or other substrate and fecal marking unne, feces, or

other substrate. All other ehmmatlons were classified as urination or defecation Maintenance and other behavior patterns. The other behaviors for which durations were recorded were grazing, resting (while standing or lying), locomotion, grooming (self or other), rolling, pawing and herding (movements which result in the movement of a female or females in a particular direction). All occurrences of vocalization were also recorded All means reported are the means of individual stallions unless otherwise indicated. Analyses of variance and correlations were done using a statistical computing program (Mlnitab, Pennsylvama State University) Single degree-of-freedom contrasts among the means to test post hoc differences were conducted using Sheffe's method (16)
RESULTS

Expertment 1. The Occurrence of Flehmen m a Semmaturahstlc Sttuatton

The Flehmen response varied w~th the estrous cycle as determined by mounting of females by the stallion; based upon the data of the two stallions with the greatest number of observed copulatlons (A and B), the Flehmen response rate increased significantly on the day of copulation (5.4 1 6) above the combined mean for all other days ( 3 . 6 0 . 2 , p<0.001, see Table 1). The mean duration of the Flehmen response was 4 . 0 - 0 . 5 (SEM) seconds. The stallions exhibited an average of 1.3 Flehmen responses per hour dunng the study, but the rate declined slgmficantly (p<0.05) throughout the study as the mares became pregnant. The rate was at its highest during the first week of the study (3 0 responses/ hour) and at Its lowest during the last week of the study in late summer (0.7 responses/hour). Based on the data of Stallions A and B this decrease in response rate correlation with a decrease in mounting behavior (r = . 81, p < 0 . 0 5 ) . The time of observation had no effect on the number or duratton of Flehmen responses or on the number of copulations performed by the two stallions (A and B) on which 4 observations per day were performed. They exhibited Flehmen 0 5 0.2 times/observation period at dawn, 0.7 0.4 at noon, 0.8 + 0.4 at sunset and 0.9 0.2 at midnight. There were no consistent differences in the sniff or Flehmen rate of stallions on pasture with different numbers of mares. See Table 2. Note that most of the sniffing was directed toward feces or toward the ground where urine may have been and most of the Flehman to unne. In 74% of the cases a Flehmen response was immediately preceded by nasal investigation (Fig. 1A). A change in orientation toward a mare, marking, or maintenance behaviors prior to the Flehmen response occurred much less frequently (Fig. lA).When Flehmen was preceded by nasal investigation, the investigation

FLEHMEN RESPONSE OF STALLIONS

1209

TABLE 2 SNIFF RATE TO VARIOUS SUBSTRATES AND FLEHMEN RATE AFTER NASAL INVESTIGATION OF VARIOUS SUBSTRATES Snlff/hr No. Mares Mare Mare Stallion In Harem Unne Feces Anogenltal Other E D C B A 2 3-4 4 13 13-18 0.33 0 0 15 0.83 0 47 4.04 0.12 1 45 0 28 1 13 0 0 26 0.26 0 20 0 32 0.67 0 28 0 47 0.17 0 52

NasalGround Nasal 0 33 1.7 4 26 1.57 2.28 0.33 0.36 0.77 0 22 0 50

Flehmen/hr No, Mares Mare Other Part Stalhon In Harem Unne Feces Anogen]tal of Mare Ground Other* E D C B A 2 3-4 4 13 13-18 033 0 09 0 13 0.06 0 14 0 0.02 0.04 0 02 0.ll 0 0 0.02 0.02 0 02 0 0 0 0 0 02 0 0.2 0 09 0.25 0.37 0 0 0.26 0 17 0 27

*Flehmen after nasal investigation of some other object or after no sexual mvestiganon

was significantly ( p < 0 . 0 1 ) m o r e often directed toward the g r o u n d , w h e r e the presence or absence o f urine could not be c o n f i r m e d , rather than to feces, u n n e or a female. O f the c h a n g e s in orientation toward a m a r e prior to the F l e h m e n r e s p o n s e , the stallion a p p r o a c h e d a m a r e m 55% o f the cases, m o u n t e d a m a r e in 25% o f the cases, herded a m a r e or m a r e s in 10% o f the cases, and left a mare in the r e m a i n i n g 10% o f the cases. Similarly, o f the m a i n t e n a n c e b e h a v i o r s occurring prior to the r e s p o n s e the stallion w a s either grazing (80% o f the cases), urinating (4%), resting while standard (8%) or w a l k i n g (8%). O f the m a r k i n g behaviors exhibited prior to the r e s p o n s e 5% were urine m a r k i n g urine, 3% were urine m a r k i n g feces a n d 9 2 % were urine m a r k i n g after sniffing the g r o u n d w h e r e no f e c e s were present a n d no urine could be observed. N o fecal m a r k i n g occurred prior to the F l e h m e n response. Sniffing the g r o u n d occurred m o r e frequently than sniffing a female or e x c r e m e n t (/9<0.005), To d e t e r m i n e w h i c h s u b s t a n c e s m o s t frequently elicited the F l e h m e n r e s p o n s e the data were weighted by calculating the percentage o f sniffs in e a c h category w h i c h were followed by F l e h m e n . T h e greatest percentage o f F l e h m e n occurred after sniffing u n n e (46%) w h i c h e x c e e d e d that o f the other percentages calculated (mean = 1 1 % , p < 0 . 0 0 5 , A N O V A ) . T h e F l e h m e n response duration after sniffing each o f the above materials did not vary significantly ( m e a n = 4 . 0 - 0 5 sec, range = 3.3-4.9). M a r k i n g was affected m o r e than other behaviors, increasing

BEHAVIOUR

PRECEDING

FLEHMEN

[3

BEHAVIOUR

FOLLOWING

FLEHMEN

II III u F VSN
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gro~'.d more

II F O
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FLEHMEN

II IIII I O I u F VSN ~OvM F

Ile/e orient towardmore

orient toword

II II L.t .,~., h.~

/

A~

L*~,,L ~ ~,~

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momlenonce behovtour

I
gr~

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UR ~ W

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RU W

mor'l--Itng behomour

31% marking behowour

I
~U

i i
tmr UMO
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:.

FW~

II

FIG 1 Behaviors immediately preceding and following the Flehmen response, expressed as percent total Each bar represents 100% and indicates the percent occurrence of each behaviour in a category. U = unne, F = feces, V = vulva, O = elsewhere on female, N = naso-nasal, F1 = flehmen, AO = approach female, LO = leave female, RU = rest upright, UR = unnate, W = walk, UMU = unne mark unne, UMF = unne mark feces, UMO = unne mark other, FMO = fecal mark other.

1210

STAHLBAUM AND HOUPT

from 13% of the behaviors preceding Flehmen to 31% of the behaviors following Flehmen (Fig. 1B). Behaviors involving a change in orientation toward the mare occurred in only 11% of the cases after Flehmen was performed. Maintenance behaviors make up 18% of the behaviors following the response The remaining 40% of the Flebmen responses were followed by further olfactory investigation of the substance Stalhons mark with unne (99%) more frequently than with feces (1%) after exhibiting Flehmen (p<0.05) Overall, during the study urine marking occurred more frequently than fecal marking (urine marking = 81%, fecal marking = 19%, p < 0 . 0 5 ) . Eight percent of unne marks were over urine, 33% were over feces and 59% occurred after sniffing and/or exhibiting Flehmen after sniffing the ground. Five percent of fecal marks were over urine, 79% over feces and 16% occurred after sniffing and/or exhibiting Flehmen over the ground. The frequency of urine marking, including bouts associated and not associated with Flehmen, was 2.52/hr; that of fecal marking 0.6/hr. Less than 9.2% of unnations and 28% of defecations were simply elimination, not marking. Of the changes in orientation after Flehmen 61% of the cases were approaches to a mare, 11% involved mounting a mare, 25% involved herding a mare or mares and 3% involved leaving a mare. Seventy-seven percent of the responses in the maintenance behavior category were followed by grazing, 15% by resting while standing, and 8% by walking with no particular orientation to a female. Responses in the nasal investigation category occurred in approximately the same percentages as those preceding the Flehmen response. In the course of the study 20 copulations were directly observed; Flehmen occurred before the copulation (within the observation period) in 13 cases. Of the 388 Flehmen responses 39 3-+9.7% (22-57%) occurred in bouts (2 or more Flehmen responses within a minute of one another).
DISCUSSION

The contrast between these results and those of MarlnIer et al. (12) who found that stallions did not Flehmen more frequently to estrous than to nonestrous unne, led us to repeat and to extend their studies on the response of stallions to eliminations. METHOD Experiment 2 Stallions were presented with a test substance (urine or feces) in a stainless steel bowl that the observer placed on the floor of their home stalls. In these behavioral bloassays the stallions were observed for ten minutes each (except in Experiment 2c which lasted 25 min). The frequency and duration of all sniffing and Flehmen responses were recorded as described below. The stallions used were sexually experienced standardbreds, thoroughbreds, or quarterhorses between the ages of three and twenty years. Within each subexperiment substances were randomly presented to each stallion. One or more control trials, in which 20 ml of distilled water was presented to the stallion, were conducted between each test trial. Each stallion was tested with only one substance per day, two to four tests per week were administered. All urine samples tested were caught from midstream voiding after administration of 500 mg furosemide intravenously to the donor animal (except in Experiment 2f) and were stored frozen and thawed immediately preceding the bloassays. Each stallion was presented with 20 ml of urine Feces were collected fresh from the stalls of the donors Each stallion was presented with one fecal ball, approximately 100 g of fecal material. The start of a Flehmen response was recorded when the upper hp hfted, and the end was recorded when the lip lowered A response was considered discrete only if the lip was lowered for at lest two seconds before it was lifted again A sniff was recorded while the stallion's nostrils were within 15 cm of the test substance A sniff was considered discrete if two seconds elapsed between two sniffing responses All means reported are the means of individual stallions. Analysis of variance and, where appropriate, paired t-tests were used to determine the significance of the differences between the means of number and duration of all sniffs and Flehmen responses. Experzment 2a. Responses to urine and water. On alternate test days eight stallions were presented with 20 ml of water and 20 ml of estrous mare urine In addition to the frequency and duration of sniffing and Flehmen, incidents of urine marking were also recorded. Experzment 2b. Responses to estrous and nonestrous urine and feces Stallions were able to distinguish urine from a nonbiological fluid so their ability to discriminate the reproductive condition of a donor female was assessed. Urine was collected twice from each of five mares, once when the mare was in estrus and once when she was in dlestrus. The reproductive state was determined by the reaction of the mares to a " t e a s e r " stallion and by rectal palpation Samples (20 ml) were presented to eight stallions by an observer blind as to the reproductive state of the mare. In addition to the frequency and duration of sniffing and Flehmen. incidents of urine marking were also recorded. Fresh feces were collected from the stalls of estrous and nonestrous mares. The stallions's responses to each type of feces was measured in separate tests Experiment 2c. Responses to vartations m volume and frequency o f urination. Because the stallions did not appear to discriminate estrous from nonestrous urine, the effect of the pattern of unnation on the Flehmen response was measured. Mares that are not in estrous urinate large volumes infrequently whereas estrous mares urinate small quantitites frequently in the presence of a stallion. To examine the difference In the response of stallions

The Flehmen response of the stalhon varies with the estrous cycle of the mares with which he is kept on pasture and occurs most frequently on the day of copulation. This implicates the Flehmen response in the sexual repertoire of the stallion The response may allow the stallion to increase his reproductive fitness by detecting estrous in mares exhibiting abnormal estrous behavior patterns, by allowing him to find estrous mares out of visual or auditory range, or by giving him the opportunity to mask estrous odors by marking and to prepare to defend estrous females in his harem before they are detected by other stallions. Although Flehmen is sometimes exhibited dunng the courtship sequence, it frequently occurs in other contexts and does not appear to be exclusively a part of courtship. In fact, the Flehmen response of the stallion is followed significantly more often by marking than by any other behavior. Similar results have been reported by Tyler (20) for New Forest ponies. The Flehmen response of the adult stallion was most frequently preceded by a sniff, and unne elicits the Flehmen response much more frequently, and consistently, than does any other material investigated. Unne has also been noted to be the substance that most frequently elicits Flehmen In horse foals (2) and by donkeys (13). The results of Experiment 1 indicate that there is no difference in rate of Flehmen with time of day, that is, the Flehmen rate was similar in all four observation periods. No great differences in behavior with time of day were expected because these horses were living in a seminaturalistic environment without supplementary feed. Their primary activity during all observation periods were grazing as has been reported previously for the mares in this study (8).

FLEHMEN RESPONSE OF STALLIONS

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TABLE 3 OLFACTORY RESPONSEOF STALLIONSTO WATER AND URINE Sniff Frequency Duration No/10 nun* Sec Responses to water and unne Responses to urine from estrous nonestrous Responses to urine from pregnant mare gelding ovanectormzed mare Responses to spontaneously voided unne furosemlde reduced unne Flehmen Frequency Duratmn No./10 nun* Sec

4.6 1 2 13.1 1.6"

5.8 - 2 3 6.8 0.9 7.0 1.0 6.6 0 8 5 8 0.8 7 3 0.8 6.2 0.6 5 1 0.5 7.7 1.0

0.3 - 0.1 10 3 -+- 1 9t 10.4 - 1.9 10.3 2.0 13.4 4 7 12639 7.8 1.7 12 4 1.2 13.6 1.6

1.3 - 0.4 6.8 +-- 1 2t 6.8 1.2 6.8 1.1 7.3 0 8 8.2+14 73 1 5 7.4 - 1.2 8.3 --- 0 8

13 4 1.5 12.8 - 1.5 16.4 _ _ _ 3.8 12.4 - 4 0 8 9 +-- 3.3 13.9 --- 1 1 16.0 --- 2 0

*These represent the mean responses of each of the stalhons. tSlgnlfiCantly greater than water (p<0 05) n = number of stallions tested.

to one large volume in comparison to many small volumes of urine, the following tests were performed: eight stallions were each presented with 100 ml of estrous mare urine in two trials each lasting 25 minutes. In one trial a single sample of 100 ml was presented in a steel bowl (single presentation). In the other trial five presentations of 20 ml of urine from same sample were made (multiple presentatmn). Dunng the single presentation trial the observer entered the stall every five minutes and moved the bowl approximately 15 cm. In the multiple presentation trial a 20 ml sample of urine in a new bowl was presented every five minutes until a total of 100 ml had been presented.

voided after she had been injected IV with 500 mg of furosemide. RESULTS

Experiment 2a. Responses to Urine and Water

The number of sniffs, Flehmen responses and urine marking episodes (urinating in a stereotyped posture described in the Method section of Experiment 1) during water control trials were significantly reduced relative to those observed during trials with estrous mare urine (Table 3). The initial sniff duration was longer if a Flehmen occurred during the ten minute test than if it did not (first sniff 8 1 sec if Flehmen occurred; fhst sniff = 5 1 sec if no Flehmen followed (p<0.05). None of the stallions urine marked water, but 5 of 8 stallions urine marked urine.

Experiment 2d. Responses to reproductive condition based on urine. Because stallions failed to discriminate estrous from nonestrous urine, their ability to distinguish gonadectomized from intact animals of either sex was determined. Five stallions were tested with 20 ml urine collected from: 1) a pregnant mare; 2) an ovariectomized mare; and 3) from a castrated male horse (gelding).

Experiment 2b. Responses to Estrous and Nonestrous Urine and Feces

The stallions did not respond differently to estrous in comparison to nonestrous mare urine. They did not show a greater olfactory response to estrous in comparison to nonestrous feces either. The stallions exhibited 10.4--.1.6 Flehmen/10 min to estrous urine and 10.3 --. 1.7 Flehmen/10 min to nonestrous urine. The mean duration was also the same (6.8 --- 1.23 to estrous and 6 . 8 1.3 to nonestrous). There was no difference in sniff rate (13.4 1.2/10 min to estrous; 12.8 1.3/10 min to nonestrous) or duration (6.6---0.9 sec to estrous; 5 . 8 2 . 7 to nonestrous). The stallions marked the estrous urine 7.3 2.7 times in the five tests and 6.1 3 . 5 in the nonestrous tests.

Experiment 2e. Responses to own and other urine and feces.

Each of six stallions were tested with: 1) their own urine; 2) the urine of another stallion; and 3) mare urine. The mean response of each stallion to estrous and diestrous urine m Experiment 2b was used as the response to mare unne. Each of eight stallions were tested with: 1) their own feces; 2) the feces of another stallion; and 3) with mare feces. The mean response of each stallion to estrous and diestrous feces in Experiment 2b was used as the response to mare feces.

Experiment 2f. Responses to normally votded from furosemideinduced urine. In experiments 2a-d furosemide (Lasix, Hoechst)
was injected into the donor animal so that a liter of isotonic urine could be collected within 30 rain. This also insured that the urine from different donors and in different reproductive states did not differ markedly m concentration. The possibility remained that the stalhon's response to furosemide-induced urine might differ from that to spontaneously voided urine. Seven stallions were tested with two 20 ml urine samples from the same mare, one sample taken during a spontaneous urination (collected from the same mare on the same day prior to the injection of diuretic); the other

Experiment 2c. Responses to Variations in Volume and Frequency of Urination

Table 4 illustrates the responses to stallions to large and small volumes or urine. The number of stuffs and Flehmen responses decreases over the 25 minutes of testing to both the large (single) and small (multiple) urine presentations; however the decrease in the number of sniffs was significantly greater to the single large

1212

STAHLBAUM AND HOUPT

2018-

.~
o

16
14-

[~1 Own Stallion

Mare

~12 o glO
Q" 8
u_
o')

stalhon's unne. They &d sniff, F(2,21)=3 57, p < 0 05 and Flehmen, F(2,21) = 6, p < 0 . 0 5 , differentially to feces They sniffed feces of mares (t= 2.65, p < 0 03) and those of other stallions ( t = 2 73, p < 0 . 0 3 ) more than their own They also exhibited Flehmen more frequently to mares' feces than to their own (t = 2.78, p < 0 . 0 3 ) or to other males (t = 2.63, p < 0 . 0 3 , Fig 2 and Fig. 3)

Experiment 2f. Discrtmmatton of Normally Voided From Furosemide-lnduced Urine

There was no difference in the number or duration of sniffs and Flehmen responses elicited by spontaneously voided urine as compared to furosemide-induced unne (Table 3) Therefore, for ease of collection, furosemide-mduced urine may be used for testing behavioral responses.
DISCUSSION

~-

4 2

Feces

Urine

FIG 2 Variation in stalhons' sniffing responses to feces and unne from themselves, from other stalhons, and from mares The bars represent the mean response of six stallions The verttcal hne represents the standard error of the mean

volume of urine (7.75 -+ 1.93) than to the multiple small volumes (3.63_ + 1.35, p < 0 025). Although the number of sniffs were greater to the large volume than to the small volume dunng the first 5 minutes of presentation, after 15 minutes only 1 stallion responded to the large volume whereas 6 responded to the third small volume presented at 15 minutes

Experiment 2d. Responses to Reproductive Conditton Based on Urine

The stallions exhibited no difference in sniff or Flehmen rate and in response to gelding, pregnant mare and ovariectomized mare urine on the basis of sniff or Flehmen rate or duration ( F < 2 0 in all cases). See Table 3.

Experiment 2e. Responses to own and Other Urine and Feces

Stalhons did not sniff or Flehmen more in response to urine from other stallions than to their own urine. They did not sniff or Flehmen more to mares' urine than to their own or another

20]
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1412 10
8-

Experiment 2 revealed that stallions sniff a 100 ml sample more frequently than a 20 ml sample presentation suggesting that they can discriminate volume of urine or the total amount of the material that stimulates olfactory invesngatlon. When the same urine was presented either as one large sample or many small samples, the stalhons habituated to it as indicated by the decrease in numbers of sniffs and Flehmen responses Nevertheless, the stallions responded significantly more to the fifth small sample presented at 20 minutes than to the large sample, i.e., they habituated more slowly to multiple small presentations. This reaction may explain why it is advantageous for mares to produce many small urinations when they are in estrus. Most of the stallion's time is spent grazing (5, 14, 20) so he is most likely to encounter a scent deposited on the ground. There will be a greater chance that the stallion will find the urine if it is deposited In several places and he wall continue to respond each time be encounters a small unne pool When the only cue available to stallions is urine, they do not discriminate, on the basis of sniffing or Flehmen, between estrous and nonestrous mares or between intact and gonadectomized horses. Stallions could discnmmate their own unne from that of other horses and could discriminate the sex of a fecal donor. Stallions are more responsive toward the feces of other stallions than toward their own. Furthermore, stallions perform Flehmen more often after investigating mare feces than after investigating their own or another stallion's. The ablhty of stallions to differentially respond toward different classes of feces suggests that fecal marks allow males to determine the sexual ~dentity of individuals which are in their vicinity Additionally, stallions are clearly able to dlscnmlnate their own feces from that of others. Turner et al. (19) as well as Feist and McCullough (5) have reported that stallions mark the feces of other horses with their own feces. Janzen (9) has speculated that horses may use the odor of feces to find their way back from unfamdiar places. Foals ingest feces, preferentially those of their mother (3,6). Taken together these findings indicate the importance of feces in individual and sex recognition in horses. GENERAL DISCUSSION The major conclusion of this study is that Flehmen by stalhons vanes with the estrous cycles of the mares in their harems, but is not part of the immediate precopulatory behavior. Furthermore, stallions can discriminate between the sexes on the barns of ehnunations, but do not appear to be able to discriminate the reproductive condition of the mare. The observation that stalhons cannot &scriminate the estrous condition of a mare based on urine alone confirms the findings of Mannier et al. (12) despite several

2 0 Feces Urine

FIG 3 Variation in stallions' Flehmen responses to feces and unne from themselves, from other stalhons and from mares. The bars represent the mean response of six stallions The vertical hne represents the standard error of the mean

FLEHMEN RESPONSE OF STALLIONS

1213

TABLE 4
EFFECT OF VARYING THE VOLUME OF URINE AND FREQUENCY OF PRESENTATION ON THE NASAL

INVESTIGATORY RESPONSESOF 8 STALLIONS

Time

0-5 mm

5-10 mm

10-15 mm Sniff number* 1.6___0.4 24 09

15-20 rmn

20-25 mm

One large (100 ml) samples of estrous unne Five small (20ml) samples of estrous unne One large (100 ml) sample of estrous unne Flvesmall(20ml) samples of estrous unne

8 8{ - 2 1 64 --- 1 7

19

---07

1.8---04 4.3 - 2 2

09--+04 40 ~ 1 6

5.0] 0.6

6 1 07 50 --- 0 9

1.6 25

Flehmen number* 1.0 0606 0 - 07 1 6 _+ 1.8

01 01 0.5 0.2

0.8 0 2

*These represent the mean of 8 stallions ].Slgmficantly more sniffs than to the larger volume. :~Slgmficantly more sniffs than the smaller volume

methodological differences between the two studtes. Based on the frequency and duration of olfactory behaviors, sniffing and Flehmen, an individual stallion can discnminate the sex, but not the estrous stage, of fecal donors and could discriminate his own feces, but not his own urine, from that of other horses The average rate of Flehmen per hour of the stallions in this study was less than that found for colts, but greater than that found for fillies and for mares (2). The rate of flehmen ts greatest m the youngest foals and decreased with age (2). As predicted, the stalhon differs from the small ruminants studied in that the rate of Flehmen peaks at the time of estrus, as indicated by copulation, rather than during diestrus as observed in goats (10) and in sheep (1). The stallion may use other cues, particularly the mare's proceptivity, to detect a receptive female and use Flehmen to synchromze hts behavior with hers. The harem stallion always has mares nearby, but young bachelor stalhons must try to locate females. These bachelor stallions may use the odor of urine or feces to determine whether a female is in the vicinity. In contrast to urine that quickly soaks into the ground, feces remain visible for many days. There is, therefore, an advantage of using feces for individual identtfication. The bachelor stallion could distinguish a solitary mare from one accompanied by a stallion, if that stallion had fecal marked on her feces. The present study indicates that stallions can differentiate the sex of the donor from unne and feces alone and that their discrimination is better when the stimulus if feces. Gtven that the Flehmen response of the stalhon varies with the

estrous cycle of the mares in his vicimty, but that the level of sexual responding does not significantly increase shortly after exhibmng the response, it ts concluded that the Flehmen response is involved in the overall monitoring of the estrous cycle. Monttorlng of the mares' estrous cycle may keep the stallion behaviorally and physiologically prepared to breed at the appropriate times yet prevent him from wastmg resources in doing so under tmproper condlUons. Ladewlg et al (10) had also hypothesized that vomeronasal stimulation maintains and stimulates a male's sexual interest. Preliminary experiments indicate that Flehmen may be followed by an increase m luteinizing hormone m the peripheral blood. Flehmen may be the mechanism whereby the stalhon's behavior is coordinated with that of the receptive mare It is possible that the stalhon is aware of the reproductive con&tlon of the mare, but does not indicate that perception by h~s olfactory behavior. An operant conditioning techmque m which the horse ~s rewarded for correct discrimination could be used to determine whether a stallion is able to make this discrimination. ACKNOWLEDGEMENTS We wish to thank K Fischer, D. Carbonaro, M O'Connell, R Panzer, L Ghcksteln, K. Dusza, D Ryan, and C Prouty for their assistance m observauons and Dr J Lowe, Dr M. Blue, Mrs M Butler, and the Comell Umverslty Equine Research Staff for their cooperation in allowing us to use their horses and facilities Additionally, we are grateful to the Harry M Zwelg Memorial Fund for Equine Research for financml support of the horses m this study

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STAHLBAUM AND HOUPT

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behavtor, seminal pH and accessory sex gland wetghts m geldings admtmstered testosterone and(or)estra&ol-1713 J Atom Scl 51 1358-1366; 1980. 19. Turner, J W , Perkins, A , Klrkpatrlck, J. F EllmmaUon marking behavior m feral horses Can J Zool. 59:1561-1566; 1981 20 Tyler, S J. The behavior and social organlzataon of the New Forest ponies, Anlm. Behav Mongr 5 87-196, 1972