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Female to female stimulation of ovarian activity in the ewe
Animal Reproduction Science 39 ( 1995) 251-258
Female to female stimulation of ovarian activity in the ewe L. Zarco”, E.F. Rodriguez, M.R.B. Angulo, J. Valencia Department0 de Reproduccibn, Facultad de Medicina Veterinaria y Zootecnia, Universidad National Autdnoma de MPxico, Apartado Postal 22-256, Mexico D.F. 14000, Mexico
Accepted 26 January 1995
Abstract
The ovulatory and oestrus responses of seasonally anovulatory ewes to the presence of ewes with synchronised oestrus was evaluated. The experiment was carried between 4 June and 1 July when the ewes were in seasonal anoestrus. Two hundred adult Suffolk and Dorset ewes were used. The animals were randomly divided into five groups balanced according to breed: Group I (treated) consisted of 25 ewes induced to cycle by the treatment for 10 days with vaginal sponges containing 40 mg of fluorogestone acetate and an injection of 200 IU of pregnant mares’ serum gonadotropin (PMSG) at the time of sponge removal. Group II (mixed) consisted of 25 untreated ewes housed in the same pen as the treated ewes throughout the experiment. Groups III, IV and V each consisted of 50 untreated ewes located in adjacent pens progressively more distant from the pen which contained the treated animals. The ewes in Group III had contact with the treated animals through the fence, while those in Groups IV and V were separated from the treated ewes by one and two pens respectively. Day 0 of the experiment was defined as the day in which the sponges were removed from the treated ewes. Blood samples for progesterone determination were obtained from 25 animals from each group on days 6, 10 and 13. Oestrus was detected twice a day using vasectomised rams introduced to each pen for 15 min in the morning and 15 min in the evening. As expected, the proportion of ewes with luteal activity was higher (P < 0.01) in the treated group than in the other four groups on days 6, 10 and 13. By day 13 progesterone levels were elevated in 87.5%, 52%, 37.5%, 32% and 13% of the ewes sampled in Groups I, II, III, IV and V respectively. There was a direct relationship between the proportion of non-treated ewes with ovarian activity and the intensity of contact with the treated ewes, being maximal in the ewes that remained mixed with the synchronised animals, and lowest in the ewes located in the most distant pen. The proportion of ewes that showed oestrus during the first 14 days after sponge removal was significantly higher in the treated (92%) and mixed (40%) groups than in Groups III ( lo%), IV (8%) and V (4%). It is concluded that the presence of a large number of ewes in oestrus can stimulate ovarian activity in seasonally anoestrous ewes. This female to female stimulation could be mediated by olfactory, visual and/or auditory stimuli. * Corresponding author. 0378-4320/95/$09.50
0 1995 Elsevier Science B.V. All rights
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L. Zarco et al. /Animal Reproduction Science 39 (199.5) 251-258
Keywords: Female effect; Biostimulation;
Pheromones
1. Introduction
Most studies regarding the stimulation of ovarian activity by social interaction in sheep have focused on the ‘male effect’, consisting of the induction of synchronised ovarian activity in anoestrous ewes by the sudden introduction of rams (Knight et al., 1983; Hall et al., 1986; Hulet et al., 1986; Cohen-Tannoudji and Signoret, 1987). In contrast, very little is known about the stimulation of ovarian activity in anoestrous ewes by the presence of other females in oestrus. Knight (1985) reported that the introduction of ewes in oestrus facilitated the onset of ovarian activity in anoestrous ewes that were stimulated to cycle by means of introduction of a male. The presence of oestrous ewes was ineffective as a mean of induction of ovarian activity in the absence of males. They therefore concluded that the role of the oestrous ewes was to stimulate the males, which in turn would exert a more effective stimulation over the anoestrous ewes. They supported this concept by demonstrating that the ram effect was more potent when the males had been mating with oestrous ewes for 2 days before they were used to stimulate anoestrous ewes. This type of male-mediated ‘female effect’ has also been found in the goat ( Walkden-Brown et al., 1993b). However, in experiments designed to test the use of melengestrol acetate for the induction of ovarian activity in sheep (Quispe, 1989) and goats (Cervantes et al., 1988), we have incidentally observed that some ( 12-20%) of the anoestrous animals in the control groups showed oestrus and ovulated simultaneously with treated animals. This happened even though males were used only to detect oestrus for periods of 15 min in the morning and 15 min in the evening, a period considered too brief for the male effect to occur (Pearce and Oldham, 1984). Furthermore, Sunderland et al. ( 1990)) working with asmall number of animals, observed that the average date of onset of ovarian activity of anoestrous ewes was advanced when they were mixed with cyclic ewes in the absence of males. Thus, it appears that there is a direct female to female stimulation of ovarian activity in sheep similar to the one recently described in goats ( Walkden-Brown et al., 1993b). The objective of the present study is to evaluate the effect of the presence of synchronised ewes on ovarian activity and oestrus behaviour of seasonally anoestrous ewes, and to determine the degree of contact needed for such an effect to occur.
2. Materials and methods The experiment was conducted at an experimental farm located close to Mexico City, at 2760 m above sea level, 19”13’N, 99”8’W. It was carried out between 4 June and 1 July, when the ewes at the farm are in seasonal anoestrus (Quispe, 1989). The ewes are housed in groups of 50 animals, each group occupying an outdoor pen of 250 m2. The pens are adjacent, forming a line of pens separated from each other by mesh fences. The males are kept in individual pens located 250 m away from the females. Two hundred adult Suffolk and Dorset ewes were used. The animals were randomly divided into five groups balanced according to breed: Group I (treated) consisted of 25
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ewes induced to cycle by the treatment for 10 days with vaginal sponges containing 40 mg of fluorogestone acetate (Chronogest, Intervet, Mexico) and an injection of 200 IU of pregnant mares’ serum gonadotropin (PMSG) (Folligon, Intervet, Mexico) at the time of sponge removal. Group II (mixed) consisted of 25 untreated ewes housed in the same pen as the treated ewes throughout the experiment. Groups III, IV and V each consisted of 50 untreated ewes located in adjacent pens progressively more distant from the pen which contained the treated animals, so that the ewes in Group III had contact with the treated animals through the fence, while those in Groups IV and V were separated from the treated animals by one and two pens (18 m and 36 m) respectively. The animals were placed in the respective pens 10 days before the sponges were inserted into the treated animals. Day 0 of the experiment was defined as the day in which the sponges were removed from the treated ewes. To evaluate whether animals had ovulated as a result of the experiment, blood samples for progesterone determination were obtained from 25 animals from each group on days 6, 10 and 13. The blood samples were obtained in heparinised tubes, the plasma was separated by centrifugation and kept frozen until assayed for progesterone using a solid phase radioimmunoassay (Srikandakumar et al., 1986; Flores et al., 1992). Progesterone levels above 1 ng ml-’ were considered indicative that ovulation had occurred. Oestrus detection started on day 0 and it was carried out twice a day using vasectomised males that were fitted with aprons to prevent copulation. A total of eight rams was used. Two rams were randomly assigned to each pen before every teasing period. The rams were introduced to each pen for 15 min in the morning and 15 min in the evening, returning to their pens between teasing periods. The order in which the pens were checked was randomly selected each time. As soon as a ewe was detected as being in oestrus it was placed in a small fenced area that was located within each pen, remaining there for the rest of the teasing period to allow the males to continue checking the other ewes. Before the start of each teasing period, the animals that had been seen in heat in previous periods were also segregated into the fenced area. Differences between groups in the number of animals ovulating and showing oestrus were analysed by x2 test.
3. Results Table 1 shows the percentage of animals from each group that had progesterone concentrations indicative of luteal activity on different days after the sponges were removed from the treated animals. As expected, the number of animals with luteal activity was significantly higher (P < 0.01) in Group I (treated) than in the other four groups on days 6, 10 and 13. The proportion of animals with luteal activity gradually declined through Groups II, III, IV and V, resulting in a direct relationship between luteal activity and the degree of contact with the treated animals. Thus, the group that was mixed with the treated ewes (Group II) had the largest proportion of animals showing luteal activity (29.2%, 37.5% and 52% on days 6, 10 and 13)) while the group located in the most distant pen (Group V) had the lowest proportion of animals with luteal activity (O%, 13% and 13% on days 6, 10 and 13). The differences between Groups II and V were significant on days 6 and 13 (P < 0.05). Table 2 presents the proportion of animals that showed oestrous behaviour on each of the first 14 days after the sponges were removed from the treated animals. The
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Table 1 Percentage of ewes with progesterone concentrations above 1 ng ml -’ in 25 ewes from each group that were sampled on different days after the sponges were removed from the treated animals
Day
6 10 13
Group” I (treated)
II (mixed)
III (adjacent)
IV (one pen distant)
V (two pens distant)
41.7a 84.0a 87.5a
29.2ab 37.5b 52.0b
4.3bc 21.7b 37.5bc
12.Obc 16.0b 32.Obc
o.oc 13.0b 13.oc
“Treated, vaginaI sponges containing fluorogestone acetate for 10 days plus 200 IU of PMSG; mixed, untreated animals kept in the same pen as the treated ones; adjacent, ewes located in the pen adjacent to that in which the treated animals were located; one and two pens distant, the number of pens that separated the animals in these groups from the treated animals. For a given day (row), values not followed by the same letter are significantly different (P < 0.05).
Table 2 Number and percentage of animals in each group that showed oestrus in each of the 14 days following removal of progestagen sponges from the treated animals
W
Group” I (treated)
II (mixed)
III (adjacent)
(n=25)
(n=25)
(n=50)
2 3 4 5 6 7 8 9 10 11 12 13 14
21(84%) _
2(8%) 1(4%)
1(2%)
Total
23(92%)a
IV (one pen distant) (n=50)
V (two pens distant) (n=50)
I
1(2%) 3(6%)
3( 12%) _ 2(8%)
1(4%) 1(4%)
1(2%) 1(2%) 1(2%)
1(2%)
1(2%) 1(2%) 1(4%)
1(4%) 10(40%)b
_ 5( lO%)c
4(8%)c
2(4%)c
“Treated, vaginal sponges containing fluorogestone acetate for 10 days plus 200 IU of PMSG; mixed, untreated animals kept in the same pen as the treated ones; adjacent, ewes located in the pen adjacent to that in which the treated animals were located; one and two pens distant, the number of pens that separated the animals in these groups from the treated animals. Different letters indicate significant differences in the proportion of animals in oestrus during the 14 days evaluated (P
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proportion of ewes that showed oestrus during this period was significantly higher in the treated group than in any other group (PcO.01). The proportion was also significantly larger (P 0.05).
4. Discussion Eighty-four percent of the animals treated with vaginal sponges showed oestrus during the second day following sponge removal, which is the expected response to this treatment (Cognie and Mauleon, 1983). All the animals that showed oestrus on day 2 in this group had progesterone levels above 1 ng ml- ’by day 10, indicating that they had ovulated. The presence of oestrus on the non-treated ewes was dispersed between days 2 and 14, but most occurred between days 2 and 7, indicating some degree of synchronisation with the treated animals. It is likely that the oestrus activity shown by the non-treated animals was induced by the presence of the treated animals, since the proportion of animals in oestrus between days 1 and 14 was significantly larger (P < 0.01) among the animals that were mixed with the treated ewes (40%) than among the ewes that were on other pens ( 10% in the adjacent pen, and 8% and 4% in the ewes that were respectively separated by one and two pens from the treated ewes) (Table 2). The rapid response of anoestrous ewes to the presence of oestrous animals suggests that there is a direct stimulation of the hypothalamo-pituitary axis similar to the one elicited by the male effect (Pearce and Oldham, 1984). The determination of progesterone concentrations demonstrated that the presence of oestrus in the non-treated ewes was not the result of imitation of behaviour, since it was accompanied by ovulation, indicating that there was induction of ovarian activity in the ewes exposed to the treated animals (Table 1). The proportion of ewes that ovulated was significantly larger in Group II than in Group V, indicating that direct contact with oestrous females induced ovarian activity. Although not significant, the proportion of ovulating ewes was also higher in Group III than in Group IV and higher in Group IV than in Group V, indicating that some degree of stimulation occurred even in the absence of direct physical contact, thus suggesting a pheromonal effect. Although visual and/or auditory stimuli cannot be discarded, it has been demonstrated in the bovine that olfactory cues are sufficient for female to female stimulation. In this regard, exposure of synchronised heifers to cervical mucus from oestrous cows resulted in enhanced oestrus synchronisation (Izard and Vandenbergh, 1982). Also, exposure to vaginal secretions from oestrous cows shortened the duration of postpartum anoestrus in beef cattle (Wright et al., 1992). The fact that the ram is able to discriminate the urine and genital secretions of oestrous ewes from those of ewes that are not in oestrus (Blissitt et al., 1990) strongly suggests the existence of a female pheromone in the ovine. However, the role of such putative pheromone remains unknown. It is not needed for oestrus detection, since anosmic rams are as efficient as intact males in the detection of oestrus (Lindsay, 1965; Gonzalez et al., 1991). Besides, although the ram is able to identify the urine of females in oestrus, the exposure to such urine does not elicit an endocrine response in the male (Gonzalez et al., 1991). In contrast,
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the physical presence of oestrous ewes induces an endocrine response in anosmic males (Gonzalez et al., 1991). If there is a female pheromone but it does not directly alter the interaction between females and males, it is tempting to speculate that the main role of such a pheromone could be female to female communication that would allow the animals in a given flock to start cycling within a short interval. This would result in a short lambing season that could offer an advantage to the flock in aspects such as defence, availability of foster mothers and flock mobility. The evolutionary advantage of this strategy is suggested by the rut behaviour of feral animals (Arnold and Dudzinski, 1978; Lincoln and Short, 1980)) in which the return of the males to the flock after a prolonged period of segregation results in a male effect that helps the females to initiate ovarian activity in a highly synchronised fashion. Once the first animals start cycling, female to female stimulation may further increase synchronisation as suggested by Walkden-Brown et al. ( 1993b). In domestic sheep, even though reproductive seasonality is mainly controlled by photoperiod (Legan and Karsch, 1980), there are other factors that modulate the response to it, so that the average date of first ovulation in a flock may vary from year to year (Robinson and Karsch, 1988; Quirke et al., 1988). However, within a given year most animals in a flock initiate ovarian activity within a few days of each other (Robinson and Karsch, 1988), which suggests the presence of social synchronising factors in the flock. The proportion of animals with luteal activity on day 13 (52%, 37.5%, 32% and 13% respectively for Groups II, III, IV and V) was always higher than the proportion of animals that showed oestrus between days 1 and 14 in the same groups (40%, lo%, 8% and 4%, respectively). Thus, the determination of progesterone levels done in this experiment showed that the induction of ovarian activity by contact with ewes in oestrus is more marked than it might appear when only oestrus behaviour is observed. These results indicate that a number of the animals ovulated without signs of oestrus, a situation that is normal during the first ovulation of the breeding season of the ewe because the expression of oestrus behaviour normally requires previous sensitisation of the nervous system by progesterone present during the preceding cycle (Karsch et al., 1980). However, it is interesting to note that 12% (21/ 175) of the animals in the non-treated groups showed oestrus even though they had not been exposed to a period of progesterone priming. Since samples for progesterone determination were not taken before day 0, the possibility that those animals could have been exposed to progesterone from a previous oestrous cycle cannot be ruled out. However, the experiment was carried out in June, and it has been previously shown that the ewes in our experimental farm do not start cycling before August (Quispe, 1989). Also, if the animals were cycling before the start of the experiment, it would have been unlikely that their natural oestrus would be synchronised with that of the treated animals. Furthermore, previous ovarian activity would not explain the differences between groups seen in this experiment. Another possibility is that the massive stimulus provided by a large number of treated ewes would have been able to elicit oestrous behaviour in some of the non-treated animals even in the absence of progesterone priming. It has been demonstrated that oestrous ewes and goats can indirectly stimulate the ovarian activity of flockmates through stimulation of the males that in turn results in an enhanced male effect (Knight et al., 1985; Walkden-Brown et al., 1993b). However, it is unlikely that the stimulation found in the present experiment was mediated through the males used to detect oestrus, since they were present in each pen for only 15 min, and sustained contact
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with males is needed for the ewes to develop follicles and ovulate (Signoret et al., 1982/ 83; Pearce and Oldham, 1984). In the goat, it was found that intermittent exposure to bucks is far less effective in inducing ovulation than continuous exposure, even when the duration of the daily exposure was much longer ( 16 h) than the 15 min used in the present experiment (Walkden-Brown et al., 1993a). Besides, exposure to the males in the present experiment was similar for all groups, with both the rams and the teasing order randomised for each teasing period. The existence of direct female to female stimulation of ovarian activity has been demonstrated in the goat ( Walkden-Brown et al., 1993b) and in the cow (Izard and Vandenbergh, 1982; Wright et al., 1992). Furthermore, Sunderland et al. (1990) used the ‘female effect’ to advance, in the absence of males, the average date of onset of the breeding season in anoestrous ewes by mixing them with ewes that were induced to cycle by changes in photoperiod. Also, O’Callaghan et al. (1994) found that, under certain conditions, the introduction of oestrous females had an effect on the ovarian activity of anoestrous ewes in the absence of males. The present study is similar to that of Sunderland et al. ( 1990)) with the exception that we used more animals and used progestagens to induce ovarian activity in a more acute and synchronised way than the induction produced with changes in photoperiod, thus we were able to observe a clearerresponse to the ‘female effect’. Knight ( 1985) was unable to show a significant stimulation of ovarian activity in anoestrous ewes when they were mixed with oestrous ewes in the absence of males. However, he observed that 9.5% of the 20 exposed ewes ovulated, compared with 0% in the group that was isolated from the oestrous ewes. Also, he did not report the proportion of oestrous ewes to those in anoestrus. The strength of the female effect might be directly related to this ratio, as a similar relationship has been demonstrated for the male effect (Reeve, 1984; Pearce and Oldham, 1984). It is possible that the lack of significance found by Knight (1985) was due to a reduced number of exposed animals and an insufficient number of oestrous females. It is concluded that the presence of a large number of ewes in oestrus can stimulate ovarian activity in anoestrous ewes, and that direct physical contact is not required for this stimulation, thus suggesting that the effect is mediated by olfactory, visual and/or auditory cues. The direct female effect could be acting together with the male effect and a malemediated female effect to improve the synchrony of reproductive activity within a flock.
References Arnold, G.W. and Dudzinski, M.L., 1978. Ethology of Free-Ranging Domestic Animals. Elsevier, Amsterdam, pp. 125-136. Blissitt, M.J., Bland, K.P. and Cottrell, D.F., 1990. Discrimination between the odours of fresh oestrous and nonoestrous ewe urine by rams. Appi. Anim. Behav. Sci., 25: 51-59. Cervantes, J., Ducoing, A., mores, G. and Zarco, L., 1988. Utilizaci6n de acetato de melengestrol y acetato de fluorogestona para la induccidn de estros en cabras prepuberes y en cabras adultas durante la estacidn de anestro. In: L. Zarco (Editor), Memorias de1 V Congreso National de la Asociaci6n de Zootecnistas y Tecnicos en Caprinocultura, 7-9 December, Mexico City, pp. 36-46. Cognie, Y. and Mauleon, P., 1983. Control of reproduction in the ewe. In: W. Haresign (Editor), Sheep Production. Butterworths, London, pp. 381-392. Cohen-Tannoudji, J. and Signoret, J.P., 1987. Effect of short exposure to the ram on later reactivity of anoestrus ewes to the male effect. Anim. Reprod. Sci., 13: 263-268.
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Flares, Cl., Amezcua, R., Zarco, L., Ducoing, A. and Quispe, T., 1992. Pregnancy diagnosis by progesterone determination on day 18 post-service in the ewe. Comparison of radioimmunoassay and enzyme immunoassay. XII Int. Congress on Animal Reproduction and Artificial Insemination, The Hague, Vol. 1, pp. 4547. Gonzalez, R., Levy, F., Orgeur, P. and Signomt, J.P., 1991. Female effect in sheep. II. Role of volatile substances from the sexually receptive female; implication of the sense of smell. Reprod. Nutr. Develop., 31: 103-109. Hall, D.G., Fogarty, N.N. and Gilmour, A.R., 1986. Seasonal&y of ovulation and e&us, and the ram effect in Poll Dorset ewes. Theriogenology, 25: 317-323. Hulet, C.V., Shupe, W.L., Ross, T. and Richards, R., 1986. Effects of nutritional environment and ram effect on breeding season in range sheep. Theriogenology, 25: 317-323. Izard, M.K. and Vandenbergh, J.G., 1982. Priming pheromones from oestrus cows increase synchronization of oestrus in dairy heifers after PGF-2 alpha injection. J. Reprod. Fertil., 66: 189-196. Karsch, F.J., Legan, S.J., Ryan, K.D. and Foster, D.L., 1980. Importance of estradiol and progesterone in regulating LH secretion and estrous behavior during the sheep estrous cycle. Biol. Reprod., 23: 404-413. Knight, T.W., 1985. Are rams necessary for the stimulation of anoestrus ewes with oestrus ewes?. Proc. N.Z. Sot. Anim. Prod., 45: 49-50. Knight, T.W., Tervit, H.R. and Lynch, P.R., 1983. Effects of boar pheromones, ram’s wool and presence of bucks on ovarian activity of anovular ewes early in the breeding season. Anim. Reprod. Sci., 6: 129-134. Legan, S.J. and Karsch, F.J., 1980. Photoperiodic control of seasonal breeding in ewes: Modulation of the negative feedback action of estradiol. Biol. Reprod., 23: 1061-1068. Lincoln, G.A. and Short, R.V., 1980. Seasonal breeding: Nature’s contraceptive. Recent Prog. Honn. Res., 36: lrn
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Lindsay, D.R., 1965. The importance of olfactory stimuli in the mating behaviour of the ram. Anim. Behav., 13: 75-78. O’Callaghan, D.O., Donovan, A., Sunderland, S.J., Boland, M.P. and Roche, J.F., 1994. Effect of the presence of male and female flockmates on reproductive activity in ewes. J. Reprod. Fertil., 100: 497-503. Pearce, D.T. and Oldham, CM., 1984. The ram effect, its mechanism and application to the management of sheep. In: D.R. Lindsay and D.T. Pearce (Editors), Reproduction in Sheep. Australian Academy of Sciences, Canberra, pp. 26-34. Quispe, Q.T., 1989. Estudios sobre el uso de1 acetato de melengestrol para la sincronizacidn e inducci6n de estros en ovejas. Tesis de Doctorado en Produccibn Animal. Universidad National Autbnoma de Mexico, 147 pp. Quirke, J.F., Stabenfeldt, G.H. and Bradford, G.E., 1988. Year and season effects on oestrus and ovarian activity in ewes of different breeds and crosses. Anim. Reprod. Sci., 16: 39-52. Reeve, J.L., 1984. Management of males for ram induced breeding of croossbred ewes in spring. In: D.R. Lindsay and D.T. Pearce (Editors), Reproduction in Sheep. Australian Academy of Sciences, Canberra, pp. 35-36. Robinson, J.E. and Karsch, F.J., 1988. Timing the breeding season of the ewe: what is the role of daylength?. Reprod. Nutr. Develop., 28: 365-374. Signoret, J.P., Fulkerson, W.J. and Lindsay, D.R., 1982/83. Effectiveness of testosterone-treated wethers and ewes as teasers. Appl. Anim. Ethol., 9: 37-45. Srikandakumar, A., Ingraham, R.H., Ellsworth, M., Archbald, L.F., Liao, A. and Godke, R.A., 1986. Comparison of a solid-phase, no extraction radioimmunoassay for progesterone with an extraction assay for monitoring luteal function in the mare, bitch and cow. Theriogenology, 26: 779-793. Sunderland, S.J., O’Callaghan, D., Boland, M.P. and Roche, J.F., 1990. Social cues can alter the time of reproductive transition in ewes. 1. Reprod. Fertil., Abstr. Ser., 5: 28. Walkden-Brown, S.W., Restall, B.J. and Henniawati, 1993a. The male effect in the Australian cashmere goat 2. Role of olfactory cues from the male. Anim. Reprod. Sci., 32: 55-67. Walkden-Brown, S.W., Restall, B.J. and Henniawati, 1993b. The male effect in the Australian cashmere goat 3. Enhancement with buck nutrition and use of oestrous females. Anim. Reprod. Sci., 32: 69-84. Wright, I.A., Rhind, S.M., Smith, A.J. and Whyte, T.K., 1992. The effect of pheromones from cows in oestrus on the duration of the post-partum anoestrous period in beef cows. Anim. Prod., 54: 465. (Abstr.)
Female to female stimulation of ovarian activity in the ewe L. Zarco”, E.F. Rodriguez, M.R.B. Angulo, J. Valencia Department0 de Reproduccibn, Facultad de Medicina Veterinaria y Zootecnia, Universidad National Autdnoma de MPxico, Apartado Postal 22-256, Mexico D.F. 14000, Mexico
Accepted 26 January 1995
Abstract
The ovulatory and oestrus responses of seasonally anovulatory ewes to the presence of ewes with synchronised oestrus was evaluated. The experiment was carried between 4 June and 1 July when the ewes were in seasonal anoestrus. Two hundred adult Suffolk and Dorset ewes were used. The animals were randomly divided into five groups balanced according to breed: Group I (treated) consisted of 25 ewes induced to cycle by the treatment for 10 days with vaginal sponges containing 40 mg of fluorogestone acetate and an injection of 200 IU of pregnant mares’ serum gonadotropin (PMSG) at the time of sponge removal. Group II (mixed) consisted of 25 untreated ewes housed in the same pen as the treated ewes throughout the experiment. Groups III, IV and V each consisted of 50 untreated ewes located in adjacent pens progressively more distant from the pen which contained the treated animals. The ewes in Group III had contact with the treated animals through the fence, while those in Groups IV and V were separated from the treated ewes by one and two pens respectively. Day 0 of the experiment was defined as the day in which the sponges were removed from the treated ewes. Blood samples for progesterone determination were obtained from 25 animals from each group on days 6, 10 and 13. Oestrus was detected twice a day using vasectomised rams introduced to each pen for 15 min in the morning and 15 min in the evening. As expected, the proportion of ewes with luteal activity was higher (P < 0.01) in the treated group than in the other four groups on days 6, 10 and 13. By day 13 progesterone levels were elevated in 87.5%, 52%, 37.5%, 32% and 13% of the ewes sampled in Groups I, II, III, IV and V respectively. There was a direct relationship between the proportion of non-treated ewes with ovarian activity and the intensity of contact with the treated ewes, being maximal in the ewes that remained mixed with the synchronised animals, and lowest in the ewes located in the most distant pen. The proportion of ewes that showed oestrus during the first 14 days after sponge removal was significantly higher in the treated (92%) and mixed (40%) groups than in Groups III ( lo%), IV (8%) and V (4%). It is concluded that the presence of a large number of ewes in oestrus can stimulate ovarian activity in seasonally anoestrous ewes. This female to female stimulation could be mediated by olfactory, visual and/or auditory stimuli. * Corresponding author. 0378-4320/95/$09.50
0 1995 Elsevier Science B.V. All rights
SSDlO378-4320(95)01384-9
reserved
252
L. Zarco et al. /Animal Reproduction Science 39 (199.5) 251-258
Keywords: Female effect; Biostimulation;
Pheromones
1. Introduction
Most studies regarding the stimulation of ovarian activity by social interaction in sheep have focused on the ‘male effect’, consisting of the induction of synchronised ovarian activity in anoestrous ewes by the sudden introduction of rams (Knight et al., 1983; Hall et al., 1986; Hulet et al., 1986; Cohen-Tannoudji and Signoret, 1987). In contrast, very little is known about the stimulation of ovarian activity in anoestrous ewes by the presence of other females in oestrus. Knight (1985) reported that the introduction of ewes in oestrus facilitated the onset of ovarian activity in anoestrous ewes that were stimulated to cycle by means of introduction of a male. The presence of oestrous ewes was ineffective as a mean of induction of ovarian activity in the absence of males. They therefore concluded that the role of the oestrous ewes was to stimulate the males, which in turn would exert a more effective stimulation over the anoestrous ewes. They supported this concept by demonstrating that the ram effect was more potent when the males had been mating with oestrous ewes for 2 days before they were used to stimulate anoestrous ewes. This type of male-mediated ‘female effect’ has also been found in the goat ( Walkden-Brown et al., 1993b). However, in experiments designed to test the use of melengestrol acetate for the induction of ovarian activity in sheep (Quispe, 1989) and goats (Cervantes et al., 1988), we have incidentally observed that some ( 12-20%) of the anoestrous animals in the control groups showed oestrus and ovulated simultaneously with treated animals. This happened even though males were used only to detect oestrus for periods of 15 min in the morning and 15 min in the evening, a period considered too brief for the male effect to occur (Pearce and Oldham, 1984). Furthermore, Sunderland et al. ( 1990)) working with asmall number of animals, observed that the average date of onset of ovarian activity of anoestrous ewes was advanced when they were mixed with cyclic ewes in the absence of males. Thus, it appears that there is a direct female to female stimulation of ovarian activity in sheep similar to the one recently described in goats ( Walkden-Brown et al., 1993b). The objective of the present study is to evaluate the effect of the presence of synchronised ewes on ovarian activity and oestrus behaviour of seasonally anoestrous ewes, and to determine the degree of contact needed for such an effect to occur.
2. Materials and methods The experiment was conducted at an experimental farm located close to Mexico City, at 2760 m above sea level, 19”13’N, 99”8’W. It was carried out between 4 June and 1 July, when the ewes at the farm are in seasonal anoestrus (Quispe, 1989). The ewes are housed in groups of 50 animals, each group occupying an outdoor pen of 250 m2. The pens are adjacent, forming a line of pens separated from each other by mesh fences. The males are kept in individual pens located 250 m away from the females. Two hundred adult Suffolk and Dorset ewes were used. The animals were randomly divided into five groups balanced according to breed: Group I (treated) consisted of 25
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ewes induced to cycle by the treatment for 10 days with vaginal sponges containing 40 mg of fluorogestone acetate (Chronogest, Intervet, Mexico) and an injection of 200 IU of pregnant mares’ serum gonadotropin (PMSG) (Folligon, Intervet, Mexico) at the time of sponge removal. Group II (mixed) consisted of 25 untreated ewes housed in the same pen as the treated ewes throughout the experiment. Groups III, IV and V each consisted of 50 untreated ewes located in adjacent pens progressively more distant from the pen which contained the treated animals, so that the ewes in Group III had contact with the treated animals through the fence, while those in Groups IV and V were separated from the treated animals by one and two pens (18 m and 36 m) respectively. The animals were placed in the respective pens 10 days before the sponges were inserted into the treated animals. Day 0 of the experiment was defined as the day in which the sponges were removed from the treated ewes. To evaluate whether animals had ovulated as a result of the experiment, blood samples for progesterone determination were obtained from 25 animals from each group on days 6, 10 and 13. The blood samples were obtained in heparinised tubes, the plasma was separated by centrifugation and kept frozen until assayed for progesterone using a solid phase radioimmunoassay (Srikandakumar et al., 1986; Flores et al., 1992). Progesterone levels above 1 ng ml-’ were considered indicative that ovulation had occurred. Oestrus detection started on day 0 and it was carried out twice a day using vasectomised males that were fitted with aprons to prevent copulation. A total of eight rams was used. Two rams were randomly assigned to each pen before every teasing period. The rams were introduced to each pen for 15 min in the morning and 15 min in the evening, returning to their pens between teasing periods. The order in which the pens were checked was randomly selected each time. As soon as a ewe was detected as being in oestrus it was placed in a small fenced area that was located within each pen, remaining there for the rest of the teasing period to allow the males to continue checking the other ewes. Before the start of each teasing period, the animals that had been seen in heat in previous periods were also segregated into the fenced area. Differences between groups in the number of animals ovulating and showing oestrus were analysed by x2 test.
3. Results Table 1 shows the percentage of animals from each group that had progesterone concentrations indicative of luteal activity on different days after the sponges were removed from the treated animals. As expected, the number of animals with luteal activity was significantly higher (P < 0.01) in Group I (treated) than in the other four groups on days 6, 10 and 13. The proportion of animals with luteal activity gradually declined through Groups II, III, IV and V, resulting in a direct relationship between luteal activity and the degree of contact with the treated animals. Thus, the group that was mixed with the treated ewes (Group II) had the largest proportion of animals showing luteal activity (29.2%, 37.5% and 52% on days 6, 10 and 13)) while the group located in the most distant pen (Group V) had the lowest proportion of animals with luteal activity (O%, 13% and 13% on days 6, 10 and 13). The differences between Groups II and V were significant on days 6 and 13 (P < 0.05). Table 2 presents the proportion of animals that showed oestrous behaviour on each of the first 14 days after the sponges were removed from the treated animals. The
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Table 1 Percentage of ewes with progesterone concentrations above 1 ng ml -’ in 25 ewes from each group that were sampled on different days after the sponges were removed from the treated animals
Day
6 10 13
Group” I (treated)
II (mixed)
III (adjacent)
IV (one pen distant)
V (two pens distant)
41.7a 84.0a 87.5a
29.2ab 37.5b 52.0b
4.3bc 21.7b 37.5bc
12.Obc 16.0b 32.Obc
o.oc 13.0b 13.oc
“Treated, vaginaI sponges containing fluorogestone acetate for 10 days plus 200 IU of PMSG; mixed, untreated animals kept in the same pen as the treated ones; adjacent, ewes located in the pen adjacent to that in which the treated animals were located; one and two pens distant, the number of pens that separated the animals in these groups from the treated animals. For a given day (row), values not followed by the same letter are significantly different (P < 0.05).
Table 2 Number and percentage of animals in each group that showed oestrus in each of the 14 days following removal of progestagen sponges from the treated animals
W
Group” I (treated)
II (mixed)
III (adjacent)
(n=25)
(n=25)
(n=50)
2 3 4 5 6 7 8 9 10 11 12 13 14
21(84%) _
2(8%) 1(4%)
1(2%)
Total
23(92%)a
IV (one pen distant) (n=50)
V (two pens distant) (n=50)
I
1(2%) 3(6%)
3( 12%) _ 2(8%)
1(4%) 1(4%)
1(2%) 1(2%) 1(2%)
1(2%)
1(2%) 1(2%) 1(4%)
1(4%) 10(40%)b
_ 5( lO%)c
4(8%)c
2(4%)c
“Treated, vaginal sponges containing fluorogestone acetate for 10 days plus 200 IU of PMSG; mixed, untreated animals kept in the same pen as the treated ones; adjacent, ewes located in the pen adjacent to that in which the treated animals were located; one and two pens distant, the number of pens that separated the animals in these groups from the treated animals. Different letters indicate significant differences in the proportion of animals in oestrus during the 14 days evaluated (P
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proportion of ewes that showed oestrus during this period was significantly higher in the treated group than in any other group (PcO.01). The proportion was also significantly larger (P
4. Discussion Eighty-four percent of the animals treated with vaginal sponges showed oestrus during the second day following sponge removal, which is the expected response to this treatment (Cognie and Mauleon, 1983). All the animals that showed oestrus on day 2 in this group had progesterone levels above 1 ng ml- ’by day 10, indicating that they had ovulated. The presence of oestrus on the non-treated ewes was dispersed between days 2 and 14, but most occurred between days 2 and 7, indicating some degree of synchronisation with the treated animals. It is likely that the oestrus activity shown by the non-treated animals was induced by the presence of the treated animals, since the proportion of animals in oestrus between days 1 and 14 was significantly larger (P < 0.01) among the animals that were mixed with the treated ewes (40%) than among the ewes that were on other pens ( 10% in the adjacent pen, and 8% and 4% in the ewes that were respectively separated by one and two pens from the treated ewes) (Table 2). The rapid response of anoestrous ewes to the presence of oestrous animals suggests that there is a direct stimulation of the hypothalamo-pituitary axis similar to the one elicited by the male effect (Pearce and Oldham, 1984). The determination of progesterone concentrations demonstrated that the presence of oestrus in the non-treated ewes was not the result of imitation of behaviour, since it was accompanied by ovulation, indicating that there was induction of ovarian activity in the ewes exposed to the treated animals (Table 1). The proportion of ewes that ovulated was significantly larger in Group II than in Group V, indicating that direct contact with oestrous females induced ovarian activity. Although not significant, the proportion of ovulating ewes was also higher in Group III than in Group IV and higher in Group IV than in Group V, indicating that some degree of stimulation occurred even in the absence of direct physical contact, thus suggesting a pheromonal effect. Although visual and/or auditory stimuli cannot be discarded, it has been demonstrated in the bovine that olfactory cues are sufficient for female to female stimulation. In this regard, exposure of synchronised heifers to cervical mucus from oestrous cows resulted in enhanced oestrus synchronisation (Izard and Vandenbergh, 1982). Also, exposure to vaginal secretions from oestrous cows shortened the duration of postpartum anoestrus in beef cattle (Wright et al., 1992). The fact that the ram is able to discriminate the urine and genital secretions of oestrous ewes from those of ewes that are not in oestrus (Blissitt et al., 1990) strongly suggests the existence of a female pheromone in the ovine. However, the role of such putative pheromone remains unknown. It is not needed for oestrus detection, since anosmic rams are as efficient as intact males in the detection of oestrus (Lindsay, 1965; Gonzalez et al., 1991). Besides, although the ram is able to identify the urine of females in oestrus, the exposure to such urine does not elicit an endocrine response in the male (Gonzalez et al., 1991). In contrast,
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the physical presence of oestrous ewes induces an endocrine response in anosmic males (Gonzalez et al., 1991). If there is a female pheromone but it does not directly alter the interaction between females and males, it is tempting to speculate that the main role of such a pheromone could be female to female communication that would allow the animals in a given flock to start cycling within a short interval. This would result in a short lambing season that could offer an advantage to the flock in aspects such as defence, availability of foster mothers and flock mobility. The evolutionary advantage of this strategy is suggested by the rut behaviour of feral animals (Arnold and Dudzinski, 1978; Lincoln and Short, 1980)) in which the return of the males to the flock after a prolonged period of segregation results in a male effect that helps the females to initiate ovarian activity in a highly synchronised fashion. Once the first animals start cycling, female to female stimulation may further increase synchronisation as suggested by Walkden-Brown et al. ( 1993b). In domestic sheep, even though reproductive seasonality is mainly controlled by photoperiod (Legan and Karsch, 1980), there are other factors that modulate the response to it, so that the average date of first ovulation in a flock may vary from year to year (Robinson and Karsch, 1988; Quirke et al., 1988). However, within a given year most animals in a flock initiate ovarian activity within a few days of each other (Robinson and Karsch, 1988), which suggests the presence of social synchronising factors in the flock. The proportion of animals with luteal activity on day 13 (52%, 37.5%, 32% and 13% respectively for Groups II, III, IV and V) was always higher than the proportion of animals that showed oestrus between days 1 and 14 in the same groups (40%, lo%, 8% and 4%, respectively). Thus, the determination of progesterone levels done in this experiment showed that the induction of ovarian activity by contact with ewes in oestrus is more marked than it might appear when only oestrus behaviour is observed. These results indicate that a number of the animals ovulated without signs of oestrus, a situation that is normal during the first ovulation of the breeding season of the ewe because the expression of oestrus behaviour normally requires previous sensitisation of the nervous system by progesterone present during the preceding cycle (Karsch et al., 1980). However, it is interesting to note that 12% (21/ 175) of the animals in the non-treated groups showed oestrus even though they had not been exposed to a period of progesterone priming. Since samples for progesterone determination were not taken before day 0, the possibility that those animals could have been exposed to progesterone from a previous oestrous cycle cannot be ruled out. However, the experiment was carried out in June, and it has been previously shown that the ewes in our experimental farm do not start cycling before August (Quispe, 1989). Also, if the animals were cycling before the start of the experiment, it would have been unlikely that their natural oestrus would be synchronised with that of the treated animals. Furthermore, previous ovarian activity would not explain the differences between groups seen in this experiment. Another possibility is that the massive stimulus provided by a large number of treated ewes would have been able to elicit oestrous behaviour in some of the non-treated animals even in the absence of progesterone priming. It has been demonstrated that oestrous ewes and goats can indirectly stimulate the ovarian activity of flockmates through stimulation of the males that in turn results in an enhanced male effect (Knight et al., 1985; Walkden-Brown et al., 1993b). However, it is unlikely that the stimulation found in the present experiment was mediated through the males used to detect oestrus, since they were present in each pen for only 15 min, and sustained contact
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with males is needed for the ewes to develop follicles and ovulate (Signoret et al., 1982/ 83; Pearce and Oldham, 1984). In the goat, it was found that intermittent exposure to bucks is far less effective in inducing ovulation than continuous exposure, even when the duration of the daily exposure was much longer ( 16 h) than the 15 min used in the present experiment (Walkden-Brown et al., 1993a). Besides, exposure to the males in the present experiment was similar for all groups, with both the rams and the teasing order randomised for each teasing period. The existence of direct female to female stimulation of ovarian activity has been demonstrated in the goat ( Walkden-Brown et al., 1993b) and in the cow (Izard and Vandenbergh, 1982; Wright et al., 1992). Furthermore, Sunderland et al. (1990) used the ‘female effect’ to advance, in the absence of males, the average date of onset of the breeding season in anoestrous ewes by mixing them with ewes that were induced to cycle by changes in photoperiod. Also, O’Callaghan et al. (1994) found that, under certain conditions, the introduction of oestrous females had an effect on the ovarian activity of anoestrous ewes in the absence of males. The present study is similar to that of Sunderland et al. ( 1990)) with the exception that we used more animals and used progestagens to induce ovarian activity in a more acute and synchronised way than the induction produced with changes in photoperiod, thus we were able to observe a clearerresponse to the ‘female effect’. Knight ( 1985) was unable to show a significant stimulation of ovarian activity in anoestrous ewes when they were mixed with oestrous ewes in the absence of males. However, he observed that 9.5% of the 20 exposed ewes ovulated, compared with 0% in the group that was isolated from the oestrous ewes. Also, he did not report the proportion of oestrous ewes to those in anoestrus. The strength of the female effect might be directly related to this ratio, as a similar relationship has been demonstrated for the male effect (Reeve, 1984; Pearce and Oldham, 1984). It is possible that the lack of significance found by Knight (1985) was due to a reduced number of exposed animals and an insufficient number of oestrous females. It is concluded that the presence of a large number of ewes in oestrus can stimulate ovarian activity in anoestrous ewes, and that direct physical contact is not required for this stimulation, thus suggesting that the effect is mediated by olfactory, visual and/or auditory cues. The direct female effect could be acting together with the male effect and a malemediated female effect to improve the synchrony of reproductive activity within a flock.
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