THE FLEHMEN RESPONSE OF BULLS AND COWS. K. A. Houpt, W. Rivera and L. Glickstein

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1 THE FLEHMEN RESPONSE OF BULLS AND COWS K. A. Houpt, W. Rivera and L. Glickstein Department of Physiology New York State College of Veterinary Medicine Cornell University Ithaca, NY Received for publication: Scp i ember 2(, 1988 Accepted: dune {, lejf: ABSTRACT The response of dairy bulls to the urine of cows in various stages of the reproductive cycle was quantified by presenting 200 ml of urine in a stainless steel bowl to the stanchioned bulls for 10 min. Estrous mucus was also presented in the same manner. Sniffs, nose licks and flehmen responses were recorded. Of 15 bulls tested only 7, or 47%, met the criteria of two flehmens in response to estrous urine. Among these bulls, the rate of flehmen was higher to estrous urine (6.1 ± 1 f1ehmen/i0 min) than to nonestrous urine (3.5 ± 0.6 flehmen/io min, paired t = 3.1, P < 0.03). F1ehmen duration was also longer in response to estrous urine (6.4 ± 0.4 sec) than to nonestrous urine (5.7 ± 0.4 sec, t = 2.65, P < 0.03). There were no significant differences between the sniff frequencies and durations or in the number of licks to estrous and diestrous urine. There were significantly more f1ehmen responses to estrous urine (7 ± 1.4/10 min) than to mucus (2.25 ± 0.9/10 min, t = 4.75, P < 0.01). The response to water (0.6 ± 0.3/10 min) was not different from that to mucus (t ~ 2.37, P < 0.10). The spontaneous flehmen rate of dairy bulls in their home stalls was 3.2 ± O. 7/24 h. Although estrous cows did not exhibit flehmen frequently, they did sniff bull urine more frequently (3.6 ± 0.6/10 min) than nonestrous cows (1.8 ± 0.3/10 min, t = 2.4, P < 0.03). Key words: bull, cow, flehmen, estrus behavior Acknowledgements We appreciate the cooperation of Eastern Artificial Insemination Cooperative in this study. We are particularly grateful for the help of Bruce Bean and Gordon Nickerson. We appreciate the cooperation of Eastern Artificial Insemination Cooperative in this study. We are particularly grateful for the help of Bruce Bean and Gordon Nickerson.

2 INTRODUCTION The function of the flehmen, or lipcurl, response in bovine reproduction remains to be elucidated. Estes (1) proposed that flehmen by male ungulates functions in discrimination of the reproductive state of the female. He further hypothesized that flehmen is involved in the transfer of material to the vomeronasal organ. Recently, Ladewig and Hart (2) have shown that, in the goat, flehmen does indeed function to transfer material from the mouth to the vomeronasal organ, but whether flehmen serves the same function in goats and whether material can be transferred from the ground to the vomeronasal organ has not been demonstrated in an ungulate. Hradecky et ale (3) described a single bull whose rate of flehmen varied with the estrous cycle of the cows to which he was exposed, peaking at the time of estrous. This is similar to the sheep (4), but not to the goat that flehmens more frequently to diestrus than to estrous females (5,6). Garcia et al. (7) noted that bulls were able to discriminate ovulatory from post ovulatory cows. The purpose of this study was to determine whether the rate of flehmen or other olfactory investigatory behavior toward cow urine varied with the reproductive state of the donor cow. In addition, we wished to determine whether cows would exhibit flehmen in response to either cow or bull urine and, if so, whether their response varied with their own estrous cycle. MATERIALS AND METHODS Urine was collected from spontaneously urinating cows. Mucus was collected directly from the vagina using a plastic gloved hand and scooped into a stainless steel bowl. Estrous was determined by an independent observer a few hours before urine collection. Urine was frozen in glass containers until use, except as noted below. Bulls were kept in pairs in a 4.55 x 3.8-m pen with access to a 29 x 4.8 -m paddock. Each bull was placed in a stock chute for the experiment. Only one bull was tested each day, and that bull was tested with only one substance. The substances tested were distilled water as a control, estrous cow urine, estrous mucus and diestrous cow urine. Two hundred milliliters of the test substance were placed in a stainless steel bowl that was placed inside a bucket attached to the chute to prevent spillage. During the lo-min tests, the frequency and duration of flehmen and sniffing (muzzle within bucket) and the occurrence of nostril licking were recorded by one person, while the other, the observer, watched the bull. Before testing began, a criterion of a mean of two flehmen responses to estrous urine was established before a bull was considered a responder. The cows were housed in a 45-cow stanchion barn. They were released into a 24 x 51-m paddock each morning. Tests on cows were conducted similarly to those on bulls except that the cows were tested in their home stanchion, and the substance (either estrous cow urine or bull urine) was presented in a stainless steel bowl without a bucket. released into a 24 x 5l-m paddock each morning. Tests on cows were conducted similarly to those on bulls except that the cows were tested in their home stanchion, and the substance (either estrous cow urine or bull urine) was presented in a stainless steel bowl without a bucket.

3 ~I:DTI:I\JlDt:D100a \/nl I)") ~In I) THERIOGENOLOGY Spontaneous Flehmen Rate Nine Holstein bulls ranging in age from 5 to 11 yr were used in the study. They were housed individually in pipe rail stalls 3.6-m wide and 3.6 to 6.1-m long and bedded on wood shavings. They were fed 3.6 to 4.5 kg of grain and 5.5 kg of hay. Semen was collected once or twice a week from eight bulls; the ninth bull was not collected at all (Table 1). A total of 48 bulls were housed in the 59 x 25-m barn. A low light camera (Panasonic CCTV-WV1460) with an 8-mm lens and a time lapse videorecorder (Panasonic NV-80S0) set to record at one sixth the normal speed were used to record the bulls' behavior 24 hid for 5 to 12 d from late May until early August. The lights were turned on at 0530 h and off at 2130 h, but one row of lights was left on to facilitate videorecording. Most of the animal handlers arrived at 0530 h and left at 1530 h. The bulls were fed at 0530 h and again at 1800 h. One person was present during the late afternoon and evening. The bulls were alone from 2130 h when the lights went off until 0500 h. One observer read all the tapes by playing them back at normal speed and all occurrences of flehmen were recorded. The behavior occurring during the minute before flehmen was also recorded. The behaviors recorded were urination, bellowing, sniff, standing, ruminating, lying, rubbing, horning (digging the head into the bedding), eating, masturbating, mouthing and drinking. The non-reproductive behavior of these bulls is reported in Houpt and Wollney (8). Statistical Analysis A paired t-test was used to determine the significance of differences in the number of flehmen responses, the number of sniff responses, the number of licks, the duration of flehmen and the duration of sniffs between estrous and nonestrous urine, between estrous urine and mucus and between mucus and water (in the case of bulls) and between the responses of cows when in estrus and when not in estrus. Spearman rank order correlations were calculated between the flehmen rate and the age of the bull, the frequency with which semen was collected and the semen characteristics including volume total sperm and sperm concentration. RESULTS Of the bulls tested only seven (47%) met the criteria of two flehmens. The rate of flehmen was higher to estrous urine (6.1 ± 1 flehmen/io min) than to nonestrous urine (3.5 ± 0.6 flehmen/io min, paired t = 3.1, P < 0.03). Six of the seven bulls responded more to estrous urine. Flehmen duration was also longer in response to estrous urine (6.4 ± 0.4 sec) than to nonestrous urine (5.7 ± 0.4 sec, t = P < 0.03). Although the means are similar, six of seven bulls had a longer duration to estrous urine. There were no significant differences between the sniff frequencies and durations, or in the number of licks to estrous and diestrous urine. Only four responder bulls were tested. There were significantly more Flehmen responses to estrous urine (7 ± 1: ~Ll-9 m.tnl..j:.bg.t.:l.t~l I1lll~..Y~_ l~._25_.±.._q.~~lq_i!ljll,- ~ _~ _~_. I~ _L_~ < Q. Qll ~ ~~ duration to estrous urine. There were no significant differences between the sniff frequencies and durations, or in the number of licks to estrous and diestrous urine. Only four responder bulls were tested. There were significantly more Flehmen responses to estrous urine (7 ± 1.4/10 min) than to mucus (2.25 ± 0.9/10 min, t = 4.75, P < 0.01). The

4 response mucus (t 7.0 c: E 6.0 a '"'- c: 5.0 Q) E..c: 4.0 (J) u:: to water (0.6 ± 0.3/10 min) was not different from that to 2.37, P < 0.10; Figure 1). Water Estrous Diestrous "" "" "" A "" A "" A "" A A A A A A A A A A A A A A A A A A A A A A A A A A A "" A A A A A A Ai "., A A A A A Mucus Figure 1. The mean (± SEM) responses of bulls to estrous and nonestrous urine, mucus and water. The frequency of f1ehmen was low in both estrous and diestrus cows. The mean flehmen rate of estrous cows to bull urine was 0.2 ± 0.2/10 min; that of nonestrous cows was 0.3 ± 0.1/10 min. Only two of the cows exhibited flehmen to bull urine while in diestrus and only five exhibited f1ehmen while in estrus. The cows did sniff bull urine significantly more often when in estrus (3.6 ± 0.6/10 min) than when in diestrus (1.8 ± 0.3/10 min, t = 2.4, P < 0.03). Sniff duration did not vary with the reproductive cycle (2.2 ± 0.4 sec estrus; 2.0 ± 0.4 sec diestrus). The mean flehmen rate of estrous cows to estrous cow urine was 0.7 ± 0.3/10 min, that of nonestrous cows was 0.2 ± 0.1/10 min. Only two of the cows exhibited f1ehmen to bull urine while in diestrus and only four exhibited f1ehmen while in estrus. Cow urine did not stimulate more sniffing by estrous cows (3.2 ± 0.4/10 min) than by diestrous cows (3.4 ± 0.5/10 min), in contrast to their responses to bull urine, to which diestrous cows were less responsive. One hundred and two spontaneous flehmen responses were observed among the nine bulls in the production center. The mean f1ehmen rate was 3.3 ± 0.7/d (Table 1). Table 2 lists the behaviors that preceded f1ehmen. Because standing or grazing preceded most flehmen, there was no indication of an odor cue, but on seven occasions the bull had just been urinating, and on several others, the bull had been licking himself or his neighbor. F1ehmen occurred infrequently in bouts of two (six times) or three (once). There was no significant correlation between flehmen rate and age of bull, frequency of collection or semen characteristics. oeen ur1nat1ng, ana on several otners, tne DULL naa Deen L1cK1ng n1msel~ or his neighbor. F1ehmen occurred infrequently in bouts of two (six times) or three (once). There was no significant correlation between flehmen rate and age of bull, frequency of collection or semen characteristics.

5 Table 1. The spontaneous Flehmen rate of dairy bulls in their home stalls Days Flehmen rate Collection Bull Observed Flehmen per 24 hours Age rate Ringer Marauder Cleitus Panda Tomorrow > 30 Grey Dawn Star Wars Glade Behavior Table 2. Behavior preceding Flehmen Behavior Frequency Behavior Frequency Standing 43 Horning 2 Grazing 13 Rubbing self 2 Standing Ruminating 10 Licking self 2 Flehmen 9 Masturbating 1 Urinating 7 Drinking 1 Bellowing 3 Mouthing 1 Eating 3 Licking neighbor 1 ~at1ng Licking neighbor 1

6 DISCUSSION Bulls, or rather a subset of bulls, can discriminate the reproductive state of the cow on the basis of her urine alone. Urine rather than mucus evoked the most responses, despite reports that mucus was attractive to bulls (9) and that other species could be taught to detect estrous on the basis of mucus (10). The bull is presumably responding to a substance filtered by the kidney and which is present in the urine, rather than to a vaginal product which would be expected to be present in both the mucus and urine. Future experiments using urine taken directly from the bladder will need to compare it with naturally voided urine to determine whether Bartholin's glands or other vaginal structures contribute to eliciting the flehmen response. Presumably, the odor of the urine carries the cue, and the cue may be either volatile or nonvolatile. The function of flehmen has been hypothesized to be the transport of nonvolatile material to the vomeronasal organ. This hypothesis has been demonstrated to be true in goats (2), a species in which the vomeronasal duct opens via the incisive duct into both the mouth and the nasal cavity. Although the situation has not been studied in bulls, presumably urine on the nostrils can run into the nasal opening of the vomeronasal organ during flehmen. Urine might run from the the lip into the incisive fissure, or urine picked up on the tongue could be deposited at the oral opening of the duct in the incisive pit. The functional anatomy of the bovine vomeronasal organ has been thoroughly investigated by Jacobs et al. (11), who hypothesized that rapid compressions of the incisive duct by the animal's tongue movements against the palate force fluid into the vomeronasal organ. Experiments similar to those of Ladewig et al. (2) in the goat should be performed in bulls to trace the route of urine into the vomeronasal organ. Flehmen may function mechanically to transfer urine, but the bull must first be stimulated to make the flebmen response. Bulls may make an initial flehmen to a novel or uriniferous substance. The rate of flehmen may increase in response to estrus urine either because stimulation of the main olfactory system leads to flehmen and/or because of a positive feedback through the accessory olfactory sytem, whereby movement of estrous urine into the vomeronasal organ leads to more flehmen responses, which in turn leads to the transfer of more urine into the vomeronasal organ. Initial stimulation of the main olfactory system appears to be necessary for stimulation of flehmen, at least in the goat (2). Whether or not the bull is conscious of the differences in urine with the cow's reproductive state should be investigated using operant conditioning procedures. This study confirms the finding of Hradecky et al. (3) that bulls flehmen more frequently to estrous urine or to estrous cows than to nonestrous urine or nonestrous cows. This study also confirms the findings of Sambraus and Waring (12) that bulls who have had no contact with cows, i. e. those used to produce semen for artificial insemination, are not very responsive to female cues, probably because they have learned that the presence of a male teaser is the cue for sexual activity. Bulls are similar to sheep (4) in that they are more responsive to estrous animals, but goats are more responsive to.l.j.1lu..j..ll5~ ~a.ulu.lclu~ U.L CLlLU WCL.L.J.U5 \.J.L.) "-UC1"- uu.j..j.i:a WlLU UC1VC llc1u UU \"oull"-c1'-""- with cows, i. e. those used to produce semen for artificial insemination, are not very responsive to female cues, probably because they have learned that the presence of a male teaser is the cue for sexual activity. Bulls are similar to sheep (4) in that they are more responsive to estrous animals, but goats are more responsive to

7 nonestrous animals and their eliminations (5). Perhaps more careful observations of courtship in free-ranging sheep, goats and cattle will reveal why one species spends more time responding to the urine of nonreceptive animals. If the function is to prevent the goat from wasting energy in pursuit of a nonreceptive and, therefore, nonfertile female, as has been hypothesized (6), goats may expend more energy in courtship than the other species. In contrast with the ability to discriminate a reproductive state displayed by some of our bulls, by bulls in other studies (3, 7) as well as in a study of Bos indicus bulls (13), Wallach and Price (14) have shown that bulls cannot discriminate estrogen-induced estral cows from untreated cows. There are two possible explanations: 1) Bulls do not react to ovariectomized estrogen-treated cows as they would to cows in natural estrus: 2) A greater proportion of the bulls tested were nondiscriminators rather than discriminators. The visual stimulus of the restrained cow is or may be stronger than the olfactory stimuli. Cows did appear to show more interest, as indicated by the increased sniff rate, to bull urine when they were in behavioral estrus. This is yet another indication, along with increased mounting and general activity and decreased feeding, of behavioral estrus (15), but it is not a particularly practical method of detection, since the increase in sniffing is small. It might be feasible to have a source of bull odor available to cows, so that those who were attracted to it could be noted as part of estrus detection. Whether cows are actively attracted to bulls or the odor of bulls has not been investigated, but estrous females of most ungulate species are attracted to the odor of males when they are in estrus (16). REFERENCES 1. Estes, R.D. reproduction. The role of the vomeronasal organ in mammalian Mammalia 36: (1972). 2. Ladewig, J. and Hart, B.L. Flehmen and vomeronasal organ function in male goats. Physiol. Behav. 24: (1980). 3. Hradecky, P., Sis, R.F. and Klemm, W.R. reactions throughout the bovine estrous 20: (1983). Distribution of f1ehmen cycle. Theriogenology 4. Bland, K.P. and Jubilan, B.M. Correlation of F1ehmen by male sheep with female behaviour and oestrus. Anim. Behav. 35: (1987). 5. Ladewig, J., Price, E.O. role in sexual behavior. and Hart, B. L. F1ehmen in male goats: Behav. Neur. BioI. 30: (1980). 6. O'Brien, P.H. feral goats. Flehmen: its occurrence and possible functions in Anim. Behav. 30: (1982). 6. O'Brien, P.H. feral goats. Flehmen: its occurrence and possible functions Anim. Behav. 30: (1982). in

8 7. Garcia, M.C., McDonnell, S.M., Kenney, Bull sexual behavior tests: stimulus App1. Anim. Behav. Sci. 16:1-10 (1986). R.M. and Osborne, H.G. cow affects performance. 8. Houpt, K.A. and Wo11ney, G. Frequency of masturbation and time budgets of dairy bulls used for semen production. Appl. Anim. Behav. Sci. In press. 9. Paleologou, A.M. Detecting oestrus in cows by a method based on bovine sex pheromones. Vet. Rec. 100: (1977). 10. Kiddy, C.A., Mitchell, D.S. and Hawk, H.W. Estrus-related odors in body fluids of dairy cows. J. Dairy Sci. 67: (1984). 11. Jacobs, V.L., Sis, R.F., Chenoweth, P.J., Klemm, W.R., Sherry, C.J., Coppock, C.E. Theriogeno1. 13: (1980). 12. Sambraus, H.H. and Waring, G.H. Der Einf1uss des Harns brunstiger Kuhe auf die Gesch1echts1ust von Stieren. Z. Saugetierkunde 40:49 54 (1975). 13. Reinhardt, V. F1ehmen, mounting and copulation among members of a semi-wild cattle herd. Anim. Behav. 31: (1983). 14. Wallach, S.J.R. and Price, E.O. Bulls fail to show preference for estrous females in serving capacity tests. J. Anim. Sci. 66: (1988). 15. Hurnick, C.V., King, G.J. and Robertson, H.A. Estrous and related behaviour in postpartem Holstein cows. Appl. Anim. Ethol. ~:55-68 (1975). 16. Houpt, K.A. and Wolski, T.R. Domestic Animal Behavior for Veterinarians and Animal Scientists. Iowa State University Press, Ames, 1982, pp