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The male effect in the Australian cashmere goat. 2. Role of olfactory cues from the male
Animal Reproduction Science, 32 (1993) 55-67
55
Elsevier Science Publishers B.V., Amsterdam
The male effect in the Australian cashmere goat. 2. Role of olfactory cues from the male* S.W. Walkden-Brown a, B.J. R e s t a l l b a n d H e n n i a w a t i a aDepartment of Agriculture, Universityof Queensland, St. Lucia, Qld. 4072, Australia bWollongbarAgricultural lnstitute, NSW Agriculture, Wollongbar,N.S. W. 2477, Australia (Accepted 7 October 1992)
ABSTRACT Walkden-Brown, S.W., Restall, B.J. and Henniawati, 1993. The male effect in the Australian cashmere goat. 2. Role of olfactory cues from the male. Anita. Reprod. Sci., 32: 55-67. Exposing seasonally anovulatory does to bucks may induce a synchronised onset of fertile reproductive cycling, the 'male effect'. The role of fleece and urine odours from the buck in mediating this effect was examined in three experiments. In each experiment, ovarian activity was assessed at laparoscopy on Days - 1 , 5 and 10 or 11 after the application of treatments. In Experiment 1 (April), more 18-month-old nuUiparous anovulatory does ovulated during 11 days of exposure to two bucks ( 18/ 18 ) than after continued isolation from bucks (0/18 ), wearing empty face masks ( 2/ 19 ), wearing face masks containingbuck fleece ( 1/ 17), or wearing face masks containing a chloroform/methanol extract of buck fleece (3 / 17 ) (P < 0.001 ). Face masks were applied for 6 hr on Days 0 and 1 and for 2 hr on Days 2-8. In Experiment 2 (March), previous sexual experience did not influence the ovulatory response of 30-month-old nulliparous does to 10 days of intermittent exposure ( 16 h day- 1) to bucks or buck fleece and urine in a mask. Overall, the proportion of does ovulating after exposure to bucks, buck fleece sprayed with urine, or to continued isolation from bucks was 7/29, 2/30 and 1/ 26, respectively. More does ovulated when allowed to run with two bucks for 16 h day- ~than in the other treatments combined (P < 0.05 ). In Experiment 3 (March) more anovulatory multiparous does ovulated during 10 days of continuous exposure to bucks (18/19 ) than during 10 days continuous exposure to buck fleece (8/20) or buck fleece and urine (7/20) (P< 0.01 ). More does ovulated in each of these treatments than in isolated controls ( 1/20) (P< 0.05). Buck fleece was hung in a bag in the pen and was rubbed daily on each doe's muzzle. Fresh buck urine was sprayed onto the bag of fleece, and onto each doe's muzzle, daily. The effect of wearing empty masks for 16 h day-1 on the response to bucks was equivocal, with significant heterogeneity between replicates (9/10 and 3/10 does ovulating). Amongst does ovulating by Day 5, more of those exposed to buck odours lapsed into an anovulatory state by Day 10, compared with those exposed to bucks ( 10/15 vs. 2/27; P< 0.001 ). We conclude that: (a) exposure to buck fleece alone may induce an ovulatory response in seasonally anovulatory does; (b) this response is attenuated in comparison with that induced by bucks, with
Correspondence to: S.W. Walkden-Brown, Animal Science Group, School of Agriculture, University of Western Australia, Nedlands, W.A. 6009, Australia. *A preliminary report on Experiment 1 was presented at the 20th Annual Meeting of the Australian Society for Reproductive Biology, Monash University, Melbourne, 25-27 September 1989. Preliminary reports on all three experiments were presented at the 5th International Conference on Goats, New Delhi, 2-8 March 1992.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0378-4320/93/$06.00
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S.W. WALKDENoBROWNET AL.
fewer does ovulating, and fewer ovulating does going on to re-ovulate; (c) the response is not enhanced by the addition of buck urine; (d) the intensity and duration of exposure to buck stimuli influences the ovulatory response obtained. Overall the data suggest that the male effect in these goats is not a simple reflex response to olfactory cues but rather a complex response involving the integration of a range of exteroeeptive stimuli from the buck.
INTRODUCTION
In sexually mature but anovulatory sheep and goats, sudden exposure to males often results in the synchronous onset of fertile reproductive cycling, the 'male effect' (Underwood et al., 1944; Shelton, 1960). In both species, the introduction of the male results in a rapid increase in luteinising hormone (LH) pulse frequency, followed by a pre-ovulatory LH surge and ovulation, usually within 2-3 days of male introduction (reviews: Martin et al., 1986; Chemineau, 1987). This ovulation is accompanied by oestrus in a variable proportion of goats (Chemineau, 1983; Restall, 1988) but is silent in the ewe (Schinckel, 1954). Olfactory cues from the male appear to be involved in mediating the male effect in both sheep and goats, as evidenced by ovulatory responses to nearby males without physical or visual contact (Watson and Radford, 1960; Shelton, 1980) and the depressed response of anosmic females to male introduction (Morgan et al., 1972; Chemineau et al., 1986). Further evidence is provided by experiments in which seasonally anovulatory females have ovulated in response to ram fleece or buck hair, rubbed directly on the muzzle or applied in a face mask (Knight and Lynch, 1980; Knight et al., 1983; Claus et al., 1990). However, the proportion of females ovulating in response to male odours varies between reports, with a response of equivalent magnitude to that induced by rams reported in only one study (Knight and Lynch, 1980). Other reports indicate that an ovulatory response to male odours alone may be considerably smaller than that induced by males (Shelton, 1980) or may not occur at all, despite full responsiveness to males (Pearce and Oldham, 1988). If olfactory cues are the primary means by which bucks induce ovulation in seasonally anovulatory does, such cues could find considerable application for standardising or potentiating the male effect under farming and research conditions. This paper reports three experiments examining the ovulatory response of seasonally anovulatory does to buck fleece and urine odours. MATERIALS AND METHODS
Location and animals All experiments were carried out at the Pearces Creek substation of the Wollongbar Agricultural Institute (28 ° 48' S, 153 ° 25' E ) using goats bred from
MALE EFFECT IN GOATS: OLFACTORY CUES FROM BUCKS
57
captured ferals. The location, the general characteristics of the experimental animals and their management have been described elsewhere (RestaU and Pattie, 1989). All experimental females had been isolated from males for at least 10 months prior to use in experiments. All bucks used were vasectomised.
Experimental design Experiment 1: ovulation in seasonally anovulatory nulliparous does exposed continuously to bucks, or intermittently to buck fleece odours in April At laparoscopy on 30 March 1988 (Day - 1 ) 43/114 sexually inexperienced nulliparous does aged 18 months had one or more corpora lutea (CL) on their ovaries. From this population, 100 does with mean ( _+SEM) liveweights of 23.1 _+0.26 kg and including 30 does with very fresh ( 1-3 days) or regressing CL were selected and allocated to the following treatments, such that each treatment contained 14 anovulatory does and six does with CL. ( 1 ) Isolated control: does were kept in continued isolation from bucks, no masks were used. (2) Masked control: does wore face masks without odours. (3) Buck fleece: does wore a mask containing buck fleece. (4) Buck fleece extract: does wore a face mask impregnated with a chloroform/methanol extract of buck fleece. (5) Bucks: does were allowed to run with two bucks. Ovarian activity was determined by laparoscopy on Days 5 and 11. Bucks ran continuously with does from Days 0 to 10 while masks were applied for 6 h d a y - ~on Days 0 and 1, and 2 h day -1 on Days 2-8. Buck fleece was changed daily for the first 2 days and on Day 6. Fleece extract (approximately 1 ml) was applied twice at 3 h intervals on the first 2 days and daily thereafter. The controls and mask treatments were located in 0.3-0.4 ha paddocks separated by 6 m laneways. The nearest males were those in the Bucks treatment, 70 m away in another small paddock and out of sight. All treatments received equivalent amounts of mustering and handling during the experiment.
Experiment 2: effect of prior sexual experience on ovulation in seasonally anovulatory nulliparous does exposed intermittently to bucks or buck fleece and urine in March At laparascopy on 3 March 1989 (Day 0) all of a group of 86 nulliparous does aged 30 months and with mean ( + SEM) liveweights of 31.3_+0.4 kg were anovulatory. Forty-three does had been isolated from bucks since weaning at 4 months of age (sexually naive), while 43 had been exposed to vasectomised bucks for 10 days in April the previous year, during which time 91% exhibited oestrus (sexually experienced). Apart from this 10 day period, all does had the same history, being run together as one group. Does from each
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S.W. WALKDEN-BROWN ET AL.
level of sexual experience were allocated to one of three levels of male exposure. ( 1 ) Isolated: does were kept in continued isolation from bucks, no masks were used. (2) Buck fleece+urine: does wore a face mask containing buck fleece sprayed with urine, for 16 h d a y - i. (3) Bucks: does were exposed to two bucks for 16 h d a y - 1. Treatments were applied from Days 0 to 9 inclusive, with ovarian activity determined by laparoscopy on Days 5 and 10. Face masks were put on at 16:00 h each day and removed at 08:00 h the next day. Fresh buck urine was sprayed onto the buck fleece pad daily as the mask were applied. The fleece was changed once during the experiment, on Day 2. Bucks were placed with the Bucks treatment group at 16:30 h and removed at 08:30 h daily. The isolated control groups were mustered daily with a dog to simulate the daily activity in the other treatments. Treatment groups were grazed in 0.2-0.4 ha paddocks. The control groups were upwind of and separated by 30 m and a hedge from the Buck fleece + urine treatments which in turn were 70 m away from, and out of sight of, the Bucks treatments. Naive and experienced subgroups for each level of male exposure were separated by 6-10 m laneways.
Experiment 3: ovulation in seasonally anovulatory multiparous does exposed continuously to bucks or buck odours in March At laparoscopy on 8 March 1990 (Day - 1 ), 20/20 does sampled at random from a population of 100 experimental multiparous does were anovulatory. The experimental does were aged 5.5-7.5 years, had a mean ( + SEM) liveweight of 34.5 ___0.5 kg, and had kidded in August/September 1989 followed by weaning in December. Does were allocated at random to ten experimental units, each consisting often does in a single 100 m z outdoor pen. The following treatments were then allocated to pens at random, each treatment being replicated in two pens. ( 1 ) Isolated: does were isolated from bucks, masks, or odours. (2) Buck fleece: does were subjected to continuous exposure to buck fleece. ( 3 ) Buck fleece + urine: does were subjected to continuous exposure to buck fleece and urine. (4) Bucks: does were allowed to run with two bucks. (5) Bucks + mask: does were allowed to run with two bucks, does wearing empty masks for 16 h day-1 (overnight). Treatments were applied from Days 0 to 9 inclusive with ovarian activity determined by laparoscopy on Days 5 and 10. Does were exposed to buck fleece by suspending a netting bag containing approximately 0.5 kg of fleece at muzzle height in the middle of the pen, and by rubbing a handful of fleece on the muzzle of each doe daily. Does were exposed to buck urine by having it sprayed onto the muzzle and the bag of fleece in the pen, daily. Does in the
MALE EFFECT IN GOATS: OLFACTORY CUES FROM BUCKS
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mask treatment had masks fitted between 16:00 and 08:00 h daily. The bucks used were selected for moderate to high serving capacity scores. Between Days 14 and 9 inclusive the experimental does received a daily supplement of approximately 1 kg per head of lucerne hay and oats in a 1 : 1 ratio. It had been previously established that in the absence of bucks this experimental regimen did not induce ovulation. -
Origin and preparation offleece, fleece extract and buck urine In all experiments, buck fleece samples were drawn from a thoroughly mixed pool of fleece from bucks (eight or more) shorn just before each experiment. Bucks are in rut during this period. In Experiment 1, fleece extract was obtained by washing 1.0 kg of fleece with 71 of a 2:1 solution of chloroform and methanol followed by rotary drying at 40°C and finally freeze drying. For experimental use, 5 g of dried material was reconstituted in 500 ml of petroleum ether and applied by spraying onto an absorbent pad on the face masks. In Experiments 2 and 3, buck urine was collected daily from three bucks maintained in metabolism cages, and pooled before use.
Face masks The face masks used were created from lightweight dog muzzles, based on the design ofZamora (1986) (Fig. 1 ). The area over the nostrils was covered by a pad of absorbent material onto which fleece extract was sprayed, or by a thin cotton sachet containing fleece, such that upon inspiration, air was drawn through the pad or sachet. The masks caused obvious discomfort for approximately 20 min when fitted for the very first time, but not thereafter.
Pol
Airtight material
w i r e trame
Fig. 1. Face mask used to apply buck fleece, urine and buck fleece extracts.
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S.W. W A L K D E N - B R O W N
ET AL.
Evaluation and classification of ovarian function The position and number of CL and large antral follicles were recorded for each ovary, and CL were aged on the basis of changing morphology as described for sheep (Oldham and Lindsay, 1980). Fresh ovulations and CL up to 4 days post ovulation were aged to within 1 day, beyond which CL were classified as mid-cycle, regressing or corpus albicans.
Statistical analysis Treatment differences in the proportion of does ovulating and falling into different ovulatory categories were analysed using appropriate X2 tests (Snedecor and Cochran, 1967 ). RESULTS
Experiment 1 There was a significant ovulatory response in does exposed to bucks, but not buck odours or empty masks ( P < 0.001 ) (Fig. 2 ). Data from 11 animals were excluded, from two because both ovaries were not fully observed at each laparoscopy, from one because of ill health, and from eight because the CL present at the commencement of the experiment persisted'throughout, precluding ovulation during the experiment. 18/18 b
100 , 80
•~
6o
o
40
0
~
3/17s
2O
2/19 s
1/17 a 0/18 a
o
Fig. 2. Experiment 1: percentage of seasonally anovulatory nulliparous 18-month-old does ovulating in response to continuous contact with bucks, or intermittent exposure to empty face masks, face masks containing buck fleece, or face masks containing a chloroform/methanol extract of buck fleece, over 11 days in April. Ratios not sharing a c o m m o n superscript are significantly different ( P < 0.001 ).
MALEEFFECTIN GOATS:OLFACTORYCUESFROM BUCKS
61
Of the 18 does able to ovulate in the Bucks treatment, 14 ovulated twice during the experiment (Days 0-5 and 6-11 ), four ovulated once only between Days 6 and 10 and four ovulated once only between Days 0 and 5. Amongst does ovulating only once between Days 0 and 5, two had large follicles (at least 8 mm in diameter) and two had only small follicles on their ovaries at Day 11. Of the 71 does able to ovulate in the remaining treatments none ovulated twice, four ovulated once only between Days 0 and 6, and two ovulated once only between Days 0 and 5 with only small follicles on the ovary by Day 11. The presence of a short-lived CL at the commencement of the experiment had no effect on the percentage of does ovulating during the experiment. In the Bucks treatment, 5/5 does with short-lived CL ovulated compared with 13/13 anovulatory does, while in the other treatments combined the ratios were 2/17 and 4/55 respectively.
Experiment 2 Results are presented in Fig. 3. Previous sexual experience had no effect on the ovulatory response to bucks or buck odours with 6/42 naive and 6/43 experienced does ovulating during the experiment. The proportion of does ovulating in response to buck fleece and urine did not differ from that of the isolated controls while significantly more does in the Bucks treatment ovulated than in the other two treatments combined (7/29 vs. 3/56; P < 0.05 ). Of the 29 does exposed to bucks, three ovulated twice during the experiment (Days 0-5 and Days 6-10), three ovulated once only between Days 6 and 10 and one ovulated once only between Days 0 and 5 but had a large follicle (over 8 mm in diameter) in one ovary by Day 10. Of the 56 does in 50
• •
Sexually naive Sexually experienced
4O e~
3/15 4/14
•~
30
o
21)
o
m 10
1/1] 0/13
1/15 1/15
Isolated
F1. + urine
Bucks
Fig. 3. Experiment 2: percentage o f sexually naive or experienced anovulatory 30-month-old nulliparous does ovulating in response to intermittent ( 16 h d a y - 1) exposure to bucks or buck fleece and urine in a face mask, for 10 days in March. Significantly more does ovulated in response to bucks than in the other treatments combined ( P < 0.05).
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S.W.WALKDEN-BROWNETAL.
the other treatments only three does ovulated, all once only between Days 0 and 5. One of these does had a large follicle on Day 10.
Experiment 3 The proportion of does ovulating in each replicate of each treatment is presented in Fig. 4. Does exposed to buck fleece exhibited a significant ovulatory response ( P < 0.05) which was not enhanced by the addition of buck urine to 18/19 ¢
100
•
Replicates
•
Mean
12/20b
80 60
s/2o b
7 / 2 0 a'b
40 20
1/2o"
0
Fig. 4. Experiment 3: percentage of seasonally anovulatory multiparous does ovulating during 10 days of continuous contact with bucks or continuous exposure to buck fleece and urine odours. One group of does running with bucks wore empty face masks for 16 h day- 1. Ratios not sharing a common superscript are significantly different ( P < 0.05 ). TABLE1 Experiment 3: pattern of ovulation in seasonally anovulatory does ovulating during 10 days exposure to bucks or buck odours in March Treatment
Controla Buck fleecea Fleece+urinea Bucksb Bucks+maskb Total
Does in each ovulatory category ~ (%)
Does ovulating/total does
1
2
3
4
0 (0) 2 (25) 2 (29) 15 (83) 9 (75) 28 (61)
0 (0) 1 (13) 0 (0) 1 (6) 0 (0) 2 (4)
1 (100) 5 (63) 5 (71) 1 (6) 1 (8) 13 (28)
0 (0) 0 (0) 0 (0) 1 (6) 2 (17) 3 (7)
1/20 8/20 7/20 18/19 12/20 46/99
I 1, Categories defined as follows: two ovulations Days 0-5 and Days 6-10; 2, single ovulation Days 0-5 followed by persistent CL; 3, single ovulation Days 0-5 followed by short-lived CL; 4, single ovulation Days 6-10. a'~l'reatment distributions amongst ovulatory categories are significantlydifferent (P< 0.05).
MALE EFFECT IN GOATS: OLFACTORY CUES FROM BUCKS
63
the stimulus, and was significantly smaller than the response to bucks ( P < 0.01 ). The effect of wearing empty masks on the ovulatory response to bucks was equivocal, with significant heterogeneity between replicates (9/10 and 3/10 does ovulating; P < 0 . 0 5 ). In a post-experimental investigation of this heterogeneity, bucks were exchanged between the two pens, anovulatory does (without masks) were introduced and these were examined for ovulation after 5 days. The heterogeneity was repeated (6/9 and 1/9 does ovulating) and found to be associated with the pens used rather than the bucks. The pattern of ovulation amongst does ovulating in response to buck odours differed significantly from that of does ovulating in response to bucks (Table 1 ). In the Bucks treatment a significantly higher proportion of does ovulating by Day 5 did so again by Day 10 (23/27 vs. 4/15; P<0.001 ) while in the odour treatments most does ovulating by Day 5 had ovaries free of CL or large follicles on Day 10 ( 10/15 vs. 2/27 in Bucks treatment; P<0.001 ). DISCUSSION In the present experiments, seasonally anovular does either did not ovulate in response to male odours (Experiments 1 and 2), or exhibited an attenuated ovulatory response in comparison with that induced by bucks (Experiment 3 ). The lack of an ovulatory response to buck odours in Experiments 1 and 2 may have been due to a number of factors. In these experiments buck odours were applied in face masks which may have exerted a direct suppressive effect on ovulation. However, there was no clear evidence of such a suppressive effect in Experiment 3 (Bucks + mask treatment), or in sheep experiments using face masks of a different design (Knight et al., 1983; Pearce and Oldham, 1988 ). The lack of response may also have been due to the low level of sexual experience amongst the experimental does in these experiments. As has been noted by Cohen-Tannoudji et al. (1986), responses to male olfactory stimuli may be learned, and therefore dependent upon previous exposure to males. In Experiment 2, does with limited sexual experience did not respond to buck odours but it is possible that their level of experience was insufficient to induce receptivity to male odours. It should be noted however, that under natural conditions in which a doe may conceive at her first mating, the amount of direct sexual contact with bucks may also be limited. The lack of response to buck odours in Experiment 2 may have also been due to seasonal unresponsiveness in the does, as suggested by the poor ovulatory response to bucks in this experiment. However, does are normally responsive at this time of the year (RestaU, 1992; Walkden-Brown et al., 1993 ) and it is more likely that the poor response to bucks was due to the intermittent nature of contact with them. This is supported by findings in sheep that exposure to rams for periods of less than 24 h markedly reduces the ovulatory response of ewes to ram introduction (Signoret et al., 1982; Oldham and Pearce, 1983 ).
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S.W. WALKDEN-BROWN ET AL.
While the factors listed above cannot be discounted, the most probable reason for the lack of response to male odours in Experiments 1 and 2 is the way in which the olfactory stimulus was applied. In these experiments, exposure to buck odours was intermittent and no visual contact with males was possible, while in Experiment 3 exposure to buck odours was continuous (fleece bag) with an intermittent strong stimulus (daily muzzle rubbing) coupled with distant visual and auditory contact with males and females in other treatments. In Experiments 1 and 2 the interval between successive applications of odour varied from 8 to 22 h, and it is possible that an endocrine response to buck odours did occur but was not sustained during the interval between applications, resulting in a failure to ovulate. This may also explain the poor ovulatory response following intermittent exposure to bucks in Experiment 2 and is consistent with reports in both sheep and goats that the ovulatory response to male introduction is influenced by the intensity of male stimuli (Edgar and Bilkey, 1963; Shelton, 1980; Signoret et al., 1982; Chemineau, 1987; Pearce and Oldham, 1988 ) and duration of exposure (Signoret et al., 1982; Oldham and Pearce, 1983; Cohen-Tannoudji and Signoret, 1987). Nevertheless, there are reports of the successful induction of ovulation in seasonally anovulatory ewes and does following short-term intermittent exposure to male fleece odours (Knight and Lynch, 1980; Knight et al., 1983; Claus et al., 1990). In sheep such results have only been reported in the Romney breed and may reflect breed or species differences in the importance of olfactory stimuli from the male, or the use of an olfactory stimulus of greater intensity than that used in the present experiments. The results in goats (Claus et al., 1990) may have been due to the short interval between buck hair applications (4 h ) and/or the use of a stronger olfactory stimulus. In this experiment buck hair was changed every 16 h and was collected only from the head region of bucks in rut, where sebaceous gland activity is concentrated during the rut (Jenkinson et al., 1967; Walkden-Brown, 1991 ). The intensity and duration of exposure to male stimuli appeared to influence not only the proportion of does ovulating, but also the timing and persistence of the ovulatory response. In Experiment 3, the ovulatory response to male odours was transitory in comparison with that induced by males, and in the other two experiments a similar trend was evident amongst the few does that ovulated in the treatments without bucks. In Experiment 1, temporary exposure to bucks some 50 m away around Day - 6 appeared to be responsible for the fact that 38% of the source flock of does was ovulating at the commencement of the experiment. In the absence of further stimulation during the experiment, most of these does lapsed into an anovulatory state while most of those exposed to bucks re-ovulated, providing further evidence for the role of the male stimulus in maintaining the ovulatory response. The attenuated ovulatory response to buck odours observed in Experiment 3 is consistent with the low to moderate ovulatory responses obtained in other
MALE EFFECT IN GOATS: OLFACTORY CUES FROM BUCKS
65
experiments in which ovulation has been induced in goats by exposure to male odours (Shelton, 1980; Claus et al., 1990). These results and the observation that rendering female Creole goats anosmic will reduce, but not eliminate, the ovulatory response to buck introduction (Chemineau et al., 1986), provide strong support for the view that the male effect in goats is mediated by a range ofexteroceptive stimuli from the male, of which odour is but one, as proposed by Shelton (1980). The situation appears to be similar in sheep as the induction of anosmia in ewes does not influence the initial LH response to ram introduction, or eliminate the ovulatory response (Morgan et al., 1972; CohenTannoudji et al., 1986). Tactile and visual cues from the ram seem to be important in Merino sheep, with more ewes ovulating in response to full contact with rams compared with fenceline separation or the provision of rams' wool in face masks (Pearce and Oldham, 1988). While the results of Experiment 3 clearly indicate a role for buck fleece odours in mediating the male effect, the same cannot be said for buck urine since the addition of urine to buck fleece had no effect on the ovulatory response in does in Experiments 2 and 3. This is consistent with the finding of Zamora (1986) that exposing seasonally anovulatory Australian cashmere does to buck urine (via face masks) in February did not induce a significant ovulatory response, and a similar finding in Romney ewes (Knight and Lynch, 1980). However, in both of these experiments there were non-significant increases in the number of females ovulating following exposure to male urine, possibly indicative of a weak influence. No such influence was evident in Experiment 3, although a weak inductive effect of urine may have been masked by the stronger stimulus of fleece odours. Overall, the results suggest that the ovulatory response to buck introduction is not a simple reflex response to male olfactory cues but rather a more complex response resulting from the integration of a range of sensory information from the buck. In such a paradigm the intensity of the different sensory stimuli provided by the buck and the duration of exposure to them would interact with the level of female responsiveness to determine the number of females responding, and the timing and persistence of the response. Further work is required to determine the conditions under which both the male stimulus and female receptivity are optimal. ACKNOWLEDGEMENTS
This work was supported by the Rural Credits Development Fund (Project NSWDA/8932 ). We thank W.A. Taylor, G. Allen, K. Fredrickson, the late D. Wallace, R.B. Lewis, K. Watson and B. Outerbridge for assistance with animal handling and management, and Dr. D. Tucker of Deakin University for advice on the preparation of fleece extracts.
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REFERENCES Chemineau, P., 1983. Effect on oestrus and ovulation of exposing Creole goats to the male at three times of the year. J. Reprod. Fertil., 67: 65-72. Chemineau, P., 1987. Possibilities for using bucks to stimulate ovarian and oestrus cycles in anovulatory goats - - a review. Livest. Prod. Sci., 17:135-147. Chemineau, P., Levy, F. and Thimonier, J., 1986. Effects ofanosmia on LH secretion, ovulation and oestrus behaviour induced by males in the anovular Creole goat. Anim. Reprod. Sci., 10: 125-132. Claus, R., Over, R. and Dehnhard, M., 1990. Effect of male odour on LH secretion and the induction of ovulation in seasonally anoestrous goats. Anim. Reprod. Sci., 22:27 - 38. Cohen-Tannoudji, J. and Signoret, J.P., 1987. Effect of short exposure to the ram on later reactivity of anoestrous ewes to the male effect. Anim. Reprod. Sci., 13: 263-268. Cohen-Tannoudji, J., Locatelli, A. and Signoret, J.P., 1986. Non pheromonal stimulation by the male of LH release in the anoestrus ewe. Physiol. Behav., 36: 921-924. Edgar, D.G. and Bilkey, E.A., 1963. The influence of rams on the onset of the breeding season in ewes. Proc. N.Z. Soc. Anim. Proc., 23: 79-87. Jenkinson, D.M., Blackburn, P.S. and Proudfoot, R., 1967. Seasonal changes in the skin glands of the goat. Br. Vet. J., 123: 541-549. Knight, T.W. and Lynch, P.R., 1980. Source of ram pheromones that stimulate ovulation in ewes. Anita. Reprod. Sci., 3: 133-136. Knight, T.W., Tervit, H.R. and Lynch, P.R., 1983. Effects of boar pheromones, rams wool, and presence of bucks on ovarian activity in anovular ewes early in the breeding season. Anim. Reprod. Sci., 6: 129-134. Martin, G.B., Oldham, C.M., Cognie, Y. and Pearce, D.T., 1986. The physiological responses of anovulatory ewes to the introduction of r a m s - a review. Livest. Prod. Sci., 15:219-247. Morgan, P.D., Arnold, G.W. and Lindsay, D.R., 1972. A note on the mating behaviour of ewes with various senses impaired. J. Reprod. Fertil., 30:151-152. Oldham, C.M. and Lindsay, D.R., 1980. Laparoscopy in the ewe: A photographic record of the ovarian activity of ewes experiencing normal or abnormal oestrus cycles. Anim. Reprod. Sci., 3: 119-124. Oldham, C.M. and Pearce, D.T., 1983. Mechanism of the "ram effect" Proc. Aust. Soc. Reprod. Biol., 15: 72. (Abstr.) Pearce, G.P. and Oldham, C.M., 1988. Importance of non-olfactory ram stimuli in mediating ram-induced ovulation in the ewe. J. Reprod. Fertil., 84: 333-339. Restall, B.J., 1988. Artificial insemination of Australian goats stimulated by the "buck effect". Proc. Aust. Soc. Anim. Prod., 17: 302-305. Restall, B.J., 1992. Seasonal variation in reproductive activity in Australian goats. Anim. Reprod. Sci., 27: 305-318. Restall, B.J. and Pattie, W.A., 1989. The inheritance of cashmere in Australian goats. 1. Characteristics of the base population and the effects of environmental factors. Livest. Prod. Sci., 21: 157-172. Schinckel, P.G., 1954. The effect of the presence of the ram on the ovarian activity of the ewe. Aust. J. Agric. Res., 5: 465-469. Shelton, M., 1960. The influence of the presence of the male on initiation ofoestrus cycling and ovulation in Angora does. J. Anim. Sci., 19: 368-375. Shelton, M., 1980. Goats: Influence of various exteroceptive factors on initiation of estrus and ovulation. Int. Goat Sheep Res., l: 156-162. Signoret, J.P., Fulkerson, W.J. and Lindsay, D.R., 1982. Effectiveness of testosterone treated wethers and ewes as teasers. Appl. Anim. Ethol., 9: 37-45. Snedecor, G.W. and Cochran, W.G., 1967. Statistical Methods. Iowa State University Press, Ames, IA, USA, pp. 228-253.
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Underwood, E.J., Shier, F.L. and Davenport, N., 1944. The breeding season of Merino, crossbred and British breed in the agricultural districts. J. Agric. West. Aust., II Set. 2:135-143. Walkden-Brown, S.W., 1991. Environmental and social influences on reproduction in Australian cashmere goats. Ph.D. Thesis, University of Queensland, Australia, 237 pp. Walkden-Brown, S.W., Restall, B.J. and Henniawati, 1993. The male effect in Australian cashmere goats: 1. Ovulation and oestrus in seasonally anovular does in the first 10 days following the introduction of bucks. Anim. Reprod. Sci., 32: 41-53. Watson, R.H. and Radford, H.M., 1960. The influence of rams on the onset of oestrus in Merino ewes in the spring. Aust. J. Agric. Res., 11: 65-71. Zamora, A.G.C., 1986. Studies on the influence of male goats on ovulation in does. M. Agric. Studies Thesis, University of Queensland.
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The male effect in the Australian cashmere goat. 2. Role of olfactory cues from the male* S.W. Walkden-Brown a, B.J. R e s t a l l b a n d H e n n i a w a t i a aDepartment of Agriculture, Universityof Queensland, St. Lucia, Qld. 4072, Australia bWollongbarAgricultural lnstitute, NSW Agriculture, Wollongbar,N.S. W. 2477, Australia (Accepted 7 October 1992)
ABSTRACT Walkden-Brown, S.W., Restall, B.J. and Henniawati, 1993. The male effect in the Australian cashmere goat. 2. Role of olfactory cues from the male. Anita. Reprod. Sci., 32: 55-67. Exposing seasonally anovulatory does to bucks may induce a synchronised onset of fertile reproductive cycling, the 'male effect'. The role of fleece and urine odours from the buck in mediating this effect was examined in three experiments. In each experiment, ovarian activity was assessed at laparoscopy on Days - 1 , 5 and 10 or 11 after the application of treatments. In Experiment 1 (April), more 18-month-old nuUiparous anovulatory does ovulated during 11 days of exposure to two bucks ( 18/ 18 ) than after continued isolation from bucks (0/18 ), wearing empty face masks ( 2/ 19 ), wearing face masks containingbuck fleece ( 1/ 17), or wearing face masks containing a chloroform/methanol extract of buck fleece (3 / 17 ) (P < 0.001 ). Face masks were applied for 6 hr on Days 0 and 1 and for 2 hr on Days 2-8. In Experiment 2 (March), previous sexual experience did not influence the ovulatory response of 30-month-old nulliparous does to 10 days of intermittent exposure ( 16 h day- 1) to bucks or buck fleece and urine in a mask. Overall, the proportion of does ovulating after exposure to bucks, buck fleece sprayed with urine, or to continued isolation from bucks was 7/29, 2/30 and 1/ 26, respectively. More does ovulated when allowed to run with two bucks for 16 h day- ~than in the other treatments combined (P < 0.05 ). In Experiment 3 (March) more anovulatory multiparous does ovulated during 10 days of continuous exposure to bucks (18/19 ) than during 10 days continuous exposure to buck fleece (8/20) or buck fleece and urine (7/20) (P< 0.01 ). More does ovulated in each of these treatments than in isolated controls ( 1/20) (P< 0.05). Buck fleece was hung in a bag in the pen and was rubbed daily on each doe's muzzle. Fresh buck urine was sprayed onto the bag of fleece, and onto each doe's muzzle, daily. The effect of wearing empty masks for 16 h day-1 on the response to bucks was equivocal, with significant heterogeneity between replicates (9/10 and 3/10 does ovulating). Amongst does ovulating by Day 5, more of those exposed to buck odours lapsed into an anovulatory state by Day 10, compared with those exposed to bucks ( 10/15 vs. 2/27; P< 0.001 ). We conclude that: (a) exposure to buck fleece alone may induce an ovulatory response in seasonally anovulatory does; (b) this response is attenuated in comparison with that induced by bucks, with
Correspondence to: S.W. Walkden-Brown, Animal Science Group, School of Agriculture, University of Western Australia, Nedlands, W.A. 6009, Australia. *A preliminary report on Experiment 1 was presented at the 20th Annual Meeting of the Australian Society for Reproductive Biology, Monash University, Melbourne, 25-27 September 1989. Preliminary reports on all three experiments were presented at the 5th International Conference on Goats, New Delhi, 2-8 March 1992.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0378-4320/93/$06.00
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fewer does ovulating, and fewer ovulating does going on to re-ovulate; (c) the response is not enhanced by the addition of buck urine; (d) the intensity and duration of exposure to buck stimuli influences the ovulatory response obtained. Overall the data suggest that the male effect in these goats is not a simple reflex response to olfactory cues but rather a complex response involving the integration of a range of exteroeeptive stimuli from the buck.
INTRODUCTION
In sexually mature but anovulatory sheep and goats, sudden exposure to males often results in the synchronous onset of fertile reproductive cycling, the 'male effect' (Underwood et al., 1944; Shelton, 1960). In both species, the introduction of the male results in a rapid increase in luteinising hormone (LH) pulse frequency, followed by a pre-ovulatory LH surge and ovulation, usually within 2-3 days of male introduction (reviews: Martin et al., 1986; Chemineau, 1987). This ovulation is accompanied by oestrus in a variable proportion of goats (Chemineau, 1983; Restall, 1988) but is silent in the ewe (Schinckel, 1954). Olfactory cues from the male appear to be involved in mediating the male effect in both sheep and goats, as evidenced by ovulatory responses to nearby males without physical or visual contact (Watson and Radford, 1960; Shelton, 1980) and the depressed response of anosmic females to male introduction (Morgan et al., 1972; Chemineau et al., 1986). Further evidence is provided by experiments in which seasonally anovulatory females have ovulated in response to ram fleece or buck hair, rubbed directly on the muzzle or applied in a face mask (Knight and Lynch, 1980; Knight et al., 1983; Claus et al., 1990). However, the proportion of females ovulating in response to male odours varies between reports, with a response of equivalent magnitude to that induced by rams reported in only one study (Knight and Lynch, 1980). Other reports indicate that an ovulatory response to male odours alone may be considerably smaller than that induced by males (Shelton, 1980) or may not occur at all, despite full responsiveness to males (Pearce and Oldham, 1988). If olfactory cues are the primary means by which bucks induce ovulation in seasonally anovulatory does, such cues could find considerable application for standardising or potentiating the male effect under farming and research conditions. This paper reports three experiments examining the ovulatory response of seasonally anovulatory does to buck fleece and urine odours. MATERIALS AND METHODS
Location and animals All experiments were carried out at the Pearces Creek substation of the Wollongbar Agricultural Institute (28 ° 48' S, 153 ° 25' E ) using goats bred from
MALE EFFECT IN GOATS: OLFACTORY CUES FROM BUCKS
57
captured ferals. The location, the general characteristics of the experimental animals and their management have been described elsewhere (RestaU and Pattie, 1989). All experimental females had been isolated from males for at least 10 months prior to use in experiments. All bucks used were vasectomised.
Experimental design Experiment 1: ovulation in seasonally anovulatory nulliparous does exposed continuously to bucks, or intermittently to buck fleece odours in April At laparoscopy on 30 March 1988 (Day - 1 ) 43/114 sexually inexperienced nulliparous does aged 18 months had one or more corpora lutea (CL) on their ovaries. From this population, 100 does with mean ( _+SEM) liveweights of 23.1 _+0.26 kg and including 30 does with very fresh ( 1-3 days) or regressing CL were selected and allocated to the following treatments, such that each treatment contained 14 anovulatory does and six does with CL. ( 1 ) Isolated control: does were kept in continued isolation from bucks, no masks were used. (2) Masked control: does wore face masks without odours. (3) Buck fleece: does wore a mask containing buck fleece. (4) Buck fleece extract: does wore a face mask impregnated with a chloroform/methanol extract of buck fleece. (5) Bucks: does were allowed to run with two bucks. Ovarian activity was determined by laparoscopy on Days 5 and 11. Bucks ran continuously with does from Days 0 to 10 while masks were applied for 6 h d a y - ~on Days 0 and 1, and 2 h day -1 on Days 2-8. Buck fleece was changed daily for the first 2 days and on Day 6. Fleece extract (approximately 1 ml) was applied twice at 3 h intervals on the first 2 days and daily thereafter. The controls and mask treatments were located in 0.3-0.4 ha paddocks separated by 6 m laneways. The nearest males were those in the Bucks treatment, 70 m away in another small paddock and out of sight. All treatments received equivalent amounts of mustering and handling during the experiment.
Experiment 2: effect of prior sexual experience on ovulation in seasonally anovulatory nulliparous does exposed intermittently to bucks or buck fleece and urine in March At laparascopy on 3 March 1989 (Day 0) all of a group of 86 nulliparous does aged 30 months and with mean ( + SEM) liveweights of 31.3_+0.4 kg were anovulatory. Forty-three does had been isolated from bucks since weaning at 4 months of age (sexually naive), while 43 had been exposed to vasectomised bucks for 10 days in April the previous year, during which time 91% exhibited oestrus (sexually experienced). Apart from this 10 day period, all does had the same history, being run together as one group. Does from each
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level of sexual experience were allocated to one of three levels of male exposure. ( 1 ) Isolated: does were kept in continued isolation from bucks, no masks were used. (2) Buck fleece+urine: does wore a face mask containing buck fleece sprayed with urine, for 16 h d a y - i. (3) Bucks: does were exposed to two bucks for 16 h d a y - 1. Treatments were applied from Days 0 to 9 inclusive, with ovarian activity determined by laparoscopy on Days 5 and 10. Face masks were put on at 16:00 h each day and removed at 08:00 h the next day. Fresh buck urine was sprayed onto the buck fleece pad daily as the mask were applied. The fleece was changed once during the experiment, on Day 2. Bucks were placed with the Bucks treatment group at 16:30 h and removed at 08:30 h daily. The isolated control groups were mustered daily with a dog to simulate the daily activity in the other treatments. Treatment groups were grazed in 0.2-0.4 ha paddocks. The control groups were upwind of and separated by 30 m and a hedge from the Buck fleece + urine treatments which in turn were 70 m away from, and out of sight of, the Bucks treatments. Naive and experienced subgroups for each level of male exposure were separated by 6-10 m laneways.
Experiment 3: ovulation in seasonally anovulatory multiparous does exposed continuously to bucks or buck odours in March At laparoscopy on 8 March 1990 (Day - 1 ), 20/20 does sampled at random from a population of 100 experimental multiparous does were anovulatory. The experimental does were aged 5.5-7.5 years, had a mean ( + SEM) liveweight of 34.5 ___0.5 kg, and had kidded in August/September 1989 followed by weaning in December. Does were allocated at random to ten experimental units, each consisting often does in a single 100 m z outdoor pen. The following treatments were then allocated to pens at random, each treatment being replicated in two pens. ( 1 ) Isolated: does were isolated from bucks, masks, or odours. (2) Buck fleece: does were subjected to continuous exposure to buck fleece. ( 3 ) Buck fleece + urine: does were subjected to continuous exposure to buck fleece and urine. (4) Bucks: does were allowed to run with two bucks. (5) Bucks + mask: does were allowed to run with two bucks, does wearing empty masks for 16 h day-1 (overnight). Treatments were applied from Days 0 to 9 inclusive with ovarian activity determined by laparoscopy on Days 5 and 10. Does were exposed to buck fleece by suspending a netting bag containing approximately 0.5 kg of fleece at muzzle height in the middle of the pen, and by rubbing a handful of fleece on the muzzle of each doe daily. Does were exposed to buck urine by having it sprayed onto the muzzle and the bag of fleece in the pen, daily. Does in the
MALE EFFECT IN GOATS: OLFACTORY CUES FROM BUCKS
59
mask treatment had masks fitted between 16:00 and 08:00 h daily. The bucks used were selected for moderate to high serving capacity scores. Between Days 14 and 9 inclusive the experimental does received a daily supplement of approximately 1 kg per head of lucerne hay and oats in a 1 : 1 ratio. It had been previously established that in the absence of bucks this experimental regimen did not induce ovulation. -
Origin and preparation offleece, fleece extract and buck urine In all experiments, buck fleece samples were drawn from a thoroughly mixed pool of fleece from bucks (eight or more) shorn just before each experiment. Bucks are in rut during this period. In Experiment 1, fleece extract was obtained by washing 1.0 kg of fleece with 71 of a 2:1 solution of chloroform and methanol followed by rotary drying at 40°C and finally freeze drying. For experimental use, 5 g of dried material was reconstituted in 500 ml of petroleum ether and applied by spraying onto an absorbent pad on the face masks. In Experiments 2 and 3, buck urine was collected daily from three bucks maintained in metabolism cages, and pooled before use.
Face masks The face masks used were created from lightweight dog muzzles, based on the design ofZamora (1986) (Fig. 1 ). The area over the nostrils was covered by a pad of absorbent material onto which fleece extract was sprayed, or by a thin cotton sachet containing fleece, such that upon inspiration, air was drawn through the pad or sachet. The masks caused obvious discomfort for approximately 20 min when fitted for the very first time, but not thereafter.
Pol
Airtight material
w i r e trame
Fig. 1. Face mask used to apply buck fleece, urine and buck fleece extracts.
60
S.W. W A L K D E N - B R O W N
ET AL.
Evaluation and classification of ovarian function The position and number of CL and large antral follicles were recorded for each ovary, and CL were aged on the basis of changing morphology as described for sheep (Oldham and Lindsay, 1980). Fresh ovulations and CL up to 4 days post ovulation were aged to within 1 day, beyond which CL were classified as mid-cycle, regressing or corpus albicans.
Statistical analysis Treatment differences in the proportion of does ovulating and falling into different ovulatory categories were analysed using appropriate X2 tests (Snedecor and Cochran, 1967 ). RESULTS
Experiment 1 There was a significant ovulatory response in does exposed to bucks, but not buck odours or empty masks ( P < 0.001 ) (Fig. 2 ). Data from 11 animals were excluded, from two because both ovaries were not fully observed at each laparoscopy, from one because of ill health, and from eight because the CL present at the commencement of the experiment persisted'throughout, precluding ovulation during the experiment. 18/18 b
100 , 80
•~
6o
o
40
0
~
3/17s
2O
2/19 s
1/17 a 0/18 a
o
Fig. 2. Experiment 1: percentage of seasonally anovulatory nulliparous 18-month-old does ovulating in response to continuous contact with bucks, or intermittent exposure to empty face masks, face masks containing buck fleece, or face masks containing a chloroform/methanol extract of buck fleece, over 11 days in April. Ratios not sharing a c o m m o n superscript are significantly different ( P < 0.001 ).
MALEEFFECTIN GOATS:OLFACTORYCUESFROM BUCKS
61
Of the 18 does able to ovulate in the Bucks treatment, 14 ovulated twice during the experiment (Days 0-5 and 6-11 ), four ovulated once only between Days 6 and 10 and four ovulated once only between Days 0 and 5. Amongst does ovulating only once between Days 0 and 5, two had large follicles (at least 8 mm in diameter) and two had only small follicles on their ovaries at Day 11. Of the 71 does able to ovulate in the remaining treatments none ovulated twice, four ovulated once only between Days 0 and 6, and two ovulated once only between Days 0 and 5 with only small follicles on the ovary by Day 11. The presence of a short-lived CL at the commencement of the experiment had no effect on the percentage of does ovulating during the experiment. In the Bucks treatment, 5/5 does with short-lived CL ovulated compared with 13/13 anovulatory does, while in the other treatments combined the ratios were 2/17 and 4/55 respectively.
Experiment 2 Results are presented in Fig. 3. Previous sexual experience had no effect on the ovulatory response to bucks or buck odours with 6/42 naive and 6/43 experienced does ovulating during the experiment. The proportion of does ovulating in response to buck fleece and urine did not differ from that of the isolated controls while significantly more does in the Bucks treatment ovulated than in the other two treatments combined (7/29 vs. 3/56; P < 0.05 ). Of the 29 does exposed to bucks, three ovulated twice during the experiment (Days 0-5 and Days 6-10), three ovulated once only between Days 6 and 10 and one ovulated once only between Days 0 and 5 but had a large follicle (over 8 mm in diameter) in one ovary by Day 10. Of the 56 does in 50
• •
Sexually naive Sexually experienced
4O e~
3/15 4/14
•~
30
o
21)
o
m 10
1/1] 0/13
1/15 1/15
Isolated
F1. + urine
Bucks
Fig. 3. Experiment 2: percentage o f sexually naive or experienced anovulatory 30-month-old nulliparous does ovulating in response to intermittent ( 16 h d a y - 1) exposure to bucks or buck fleece and urine in a face mask, for 10 days in March. Significantly more does ovulated in response to bucks than in the other treatments combined ( P < 0.05).
62
S.W.WALKDEN-BROWNETAL.
the other treatments only three does ovulated, all once only between Days 0 and 5. One of these does had a large follicle on Day 10.
Experiment 3 The proportion of does ovulating in each replicate of each treatment is presented in Fig. 4. Does exposed to buck fleece exhibited a significant ovulatory response ( P < 0.05) which was not enhanced by the addition of buck urine to 18/19 ¢
100
•
Replicates
•
Mean
12/20b
80 60
s/2o b
7 / 2 0 a'b
40 20
1/2o"
0
Fig. 4. Experiment 3: percentage of seasonally anovulatory multiparous does ovulating during 10 days of continuous contact with bucks or continuous exposure to buck fleece and urine odours. One group of does running with bucks wore empty face masks for 16 h day- 1. Ratios not sharing a common superscript are significantly different ( P < 0.05 ). TABLE1 Experiment 3: pattern of ovulation in seasonally anovulatory does ovulating during 10 days exposure to bucks or buck odours in March Treatment
Controla Buck fleecea Fleece+urinea Bucksb Bucks+maskb Total
Does in each ovulatory category ~ (%)
Does ovulating/total does
1
2
3
4
0 (0) 2 (25) 2 (29) 15 (83) 9 (75) 28 (61)
0 (0) 1 (13) 0 (0) 1 (6) 0 (0) 2 (4)
1 (100) 5 (63) 5 (71) 1 (6) 1 (8) 13 (28)
0 (0) 0 (0) 0 (0) 1 (6) 2 (17) 3 (7)
1/20 8/20 7/20 18/19 12/20 46/99
I 1, Categories defined as follows: two ovulations Days 0-5 and Days 6-10; 2, single ovulation Days 0-5 followed by persistent CL; 3, single ovulation Days 0-5 followed by short-lived CL; 4, single ovulation Days 6-10. a'~l'reatment distributions amongst ovulatory categories are significantlydifferent (P< 0.05).
MALE EFFECT IN GOATS: OLFACTORY CUES FROM BUCKS
63
the stimulus, and was significantly smaller than the response to bucks ( P < 0.01 ). The effect of wearing empty masks on the ovulatory response to bucks was equivocal, with significant heterogeneity between replicates (9/10 and 3/10 does ovulating; P < 0 . 0 5 ). In a post-experimental investigation of this heterogeneity, bucks were exchanged between the two pens, anovulatory does (without masks) were introduced and these were examined for ovulation after 5 days. The heterogeneity was repeated (6/9 and 1/9 does ovulating) and found to be associated with the pens used rather than the bucks. The pattern of ovulation amongst does ovulating in response to buck odours differed significantly from that of does ovulating in response to bucks (Table 1 ). In the Bucks treatment a significantly higher proportion of does ovulating by Day 5 did so again by Day 10 (23/27 vs. 4/15; P<0.001 ) while in the odour treatments most does ovulating by Day 5 had ovaries free of CL or large follicles on Day 10 ( 10/15 vs. 2/27 in Bucks treatment; P<0.001 ). DISCUSSION In the present experiments, seasonally anovular does either did not ovulate in response to male odours (Experiments 1 and 2), or exhibited an attenuated ovulatory response in comparison with that induced by bucks (Experiment 3 ). The lack of an ovulatory response to buck odours in Experiments 1 and 2 may have been due to a number of factors. In these experiments buck odours were applied in face masks which may have exerted a direct suppressive effect on ovulation. However, there was no clear evidence of such a suppressive effect in Experiment 3 (Bucks + mask treatment), or in sheep experiments using face masks of a different design (Knight et al., 1983; Pearce and Oldham, 1988 ). The lack of response may also have been due to the low level of sexual experience amongst the experimental does in these experiments. As has been noted by Cohen-Tannoudji et al. (1986), responses to male olfactory stimuli may be learned, and therefore dependent upon previous exposure to males. In Experiment 2, does with limited sexual experience did not respond to buck odours but it is possible that their level of experience was insufficient to induce receptivity to male odours. It should be noted however, that under natural conditions in which a doe may conceive at her first mating, the amount of direct sexual contact with bucks may also be limited. The lack of response to buck odours in Experiment 2 may have also been due to seasonal unresponsiveness in the does, as suggested by the poor ovulatory response to bucks in this experiment. However, does are normally responsive at this time of the year (RestaU, 1992; Walkden-Brown et al., 1993 ) and it is more likely that the poor response to bucks was due to the intermittent nature of contact with them. This is supported by findings in sheep that exposure to rams for periods of less than 24 h markedly reduces the ovulatory response of ewes to ram introduction (Signoret et al., 1982; Oldham and Pearce, 1983 ).
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S.W. WALKDEN-BROWN ET AL.
While the factors listed above cannot be discounted, the most probable reason for the lack of response to male odours in Experiments 1 and 2 is the way in which the olfactory stimulus was applied. In these experiments, exposure to buck odours was intermittent and no visual contact with males was possible, while in Experiment 3 exposure to buck odours was continuous (fleece bag) with an intermittent strong stimulus (daily muzzle rubbing) coupled with distant visual and auditory contact with males and females in other treatments. In Experiments 1 and 2 the interval between successive applications of odour varied from 8 to 22 h, and it is possible that an endocrine response to buck odours did occur but was not sustained during the interval between applications, resulting in a failure to ovulate. This may also explain the poor ovulatory response following intermittent exposure to bucks in Experiment 2 and is consistent with reports in both sheep and goats that the ovulatory response to male introduction is influenced by the intensity of male stimuli (Edgar and Bilkey, 1963; Shelton, 1980; Signoret et al., 1982; Chemineau, 1987; Pearce and Oldham, 1988 ) and duration of exposure (Signoret et al., 1982; Oldham and Pearce, 1983; Cohen-Tannoudji and Signoret, 1987). Nevertheless, there are reports of the successful induction of ovulation in seasonally anovulatory ewes and does following short-term intermittent exposure to male fleece odours (Knight and Lynch, 1980; Knight et al., 1983; Claus et al., 1990). In sheep such results have only been reported in the Romney breed and may reflect breed or species differences in the importance of olfactory stimuli from the male, or the use of an olfactory stimulus of greater intensity than that used in the present experiments. The results in goats (Claus et al., 1990) may have been due to the short interval between buck hair applications (4 h ) and/or the use of a stronger olfactory stimulus. In this experiment buck hair was changed every 16 h and was collected only from the head region of bucks in rut, where sebaceous gland activity is concentrated during the rut (Jenkinson et al., 1967; Walkden-Brown, 1991 ). The intensity and duration of exposure to male stimuli appeared to influence not only the proportion of does ovulating, but also the timing and persistence of the ovulatory response. In Experiment 3, the ovulatory response to male odours was transitory in comparison with that induced by males, and in the other two experiments a similar trend was evident amongst the few does that ovulated in the treatments without bucks. In Experiment 1, temporary exposure to bucks some 50 m away around Day - 6 appeared to be responsible for the fact that 38% of the source flock of does was ovulating at the commencement of the experiment. In the absence of further stimulation during the experiment, most of these does lapsed into an anovulatory state while most of those exposed to bucks re-ovulated, providing further evidence for the role of the male stimulus in maintaining the ovulatory response. The attenuated ovulatory response to buck odours observed in Experiment 3 is consistent with the low to moderate ovulatory responses obtained in other
MALE EFFECT IN GOATS: OLFACTORY CUES FROM BUCKS
65
experiments in which ovulation has been induced in goats by exposure to male odours (Shelton, 1980; Claus et al., 1990). These results and the observation that rendering female Creole goats anosmic will reduce, but not eliminate, the ovulatory response to buck introduction (Chemineau et al., 1986), provide strong support for the view that the male effect in goats is mediated by a range ofexteroceptive stimuli from the male, of which odour is but one, as proposed by Shelton (1980). The situation appears to be similar in sheep as the induction of anosmia in ewes does not influence the initial LH response to ram introduction, or eliminate the ovulatory response (Morgan et al., 1972; CohenTannoudji et al., 1986). Tactile and visual cues from the ram seem to be important in Merino sheep, with more ewes ovulating in response to full contact with rams compared with fenceline separation or the provision of rams' wool in face masks (Pearce and Oldham, 1988). While the results of Experiment 3 clearly indicate a role for buck fleece odours in mediating the male effect, the same cannot be said for buck urine since the addition of urine to buck fleece had no effect on the ovulatory response in does in Experiments 2 and 3. This is consistent with the finding of Zamora (1986) that exposing seasonally anovulatory Australian cashmere does to buck urine (via face masks) in February did not induce a significant ovulatory response, and a similar finding in Romney ewes (Knight and Lynch, 1980). However, in both of these experiments there were non-significant increases in the number of females ovulating following exposure to male urine, possibly indicative of a weak influence. No such influence was evident in Experiment 3, although a weak inductive effect of urine may have been masked by the stronger stimulus of fleece odours. Overall, the results suggest that the ovulatory response to buck introduction is not a simple reflex response to male olfactory cues but rather a more complex response resulting from the integration of a range of sensory information from the buck. In such a paradigm the intensity of the different sensory stimuli provided by the buck and the duration of exposure to them would interact with the level of female responsiveness to determine the number of females responding, and the timing and persistence of the response. Further work is required to determine the conditions under which both the male stimulus and female receptivity are optimal. ACKNOWLEDGEMENTS
This work was supported by the Rural Credits Development Fund (Project NSWDA/8932 ). We thank W.A. Taylor, G. Allen, K. Fredrickson, the late D. Wallace, R.B. Lewis, K. Watson and B. Outerbridge for assistance with animal handling and management, and Dr. D. Tucker of Deakin University for advice on the preparation of fleece extracts.
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