- Email: [email protected]
The introduction of rams induces an increase in pulsatile LH secretion in cyclic ewes during the breeding season
Theriogenology 68 (2007) 56–66 www.theriojournal.com
The introduction of rams induces an increase in pulsatile LH secretion in cyclic ewes during the breeding season P.A.R. Hawken a,b,*, A.P. Beard b, T. Esmaili a, H. Kadokawa c, A.C.O. Evans d, D. Blache a, G.B. Martin a a School of Animal Biology, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia School of Agriculture, Food and Rural Development, University of Newcastle, Newcastle upon Tyne NE1 7RU, United Kingdom c Department of Veterinary Medicine, Faculty of Agricultural Science, The University of Yamaguchi, Yoshida 1677-1, Yamaguchi 753-8515, Japan d School of Agriculture, Food Science and Veterinary Medicine, Conway Institute for Biomolecular and Biomedical Research, College of Life Science, University College Dublin, Belfield, Dublin 4, Ireland b
Received 5 February 2007; accepted 17 March 2007
Abstract Application of the ram effect during the breeding season has been previously disregarded because the ewe reproductive axis is powerfully inhibited by luteal phase progesterone concentrations. However, anovulatory ewes treated with exogenous progestagens respond to ram introduction with an increase in LH concentrations. We therefore tested whether cyclic ewes would respond to ram introduction with an increase in pulsatile LH secretion at all stages of the estrous cycle. We did two experiments using genotypes native to temperate or Mediterranean regions. In Experiment 1 (UK), 12 randomly cycling, North of England Mule ewes were introduced to rams midway through a frequent blood-sampling regime. Ewes in the early (EL; n = 6) and late luteal (LL; n = 6) phase responded to ram introduction with an increase in LH pulse frequency and mean and basal concentrations of LH (at least P < 0.05). In Experiment 2 (Australia), the cycles of 32 Merino ewes were synchronised using intravaginal progestagen pessaries. Pessary insertion was staggered to produce eight ewes at each stage of the estrous cycle: follicular (F), early luteal (EL), mid-luteal (ML) and late luteal (LL). In all stages of the cycle, ewes responded to ram introduction with an increase in LH pulse frequency (P < 0.01); EL, ML and LL ewes also had an increase in mean LH concentration (P < 0.05). In conclusion, ram introduction to cyclic ewes stimulated an increase in pulsatile LH secretion, independent of ewe genotype or stage of the estrous cycle. Crown Copyright # 2007 Published by Elsevier Inc. All rights reserved. Keywords: Ram effect; Breeding season; Estrous cycle; Ewes; Luteinising hormone
1. Introduction The ram effect is a well-established phenomenon that induces ovulation in anovulatory ewes. In brief, the introduction of rams stimulates an increase in pulsatile
* Corresponding author at: School of Animal Biology M085, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Tel.: +61 8 6488 3588; fax: +61 8 6488 1040. E-mail address: [email protected] (P.A.R. Hawken).
secretion of LH within minutes and, in some breeds, this is sufficient to induce a pre-ovulatory LH surge, ovulation and, subsequently, estrus and conception [1–3]. One of the subsidiary benefits is that the response to the ram effect is synchronous among the flock so it can be used as a non-pharmacological alternative to conventional methods of estrus synchronisation, an important issue in today’s consumer-driven climate [4]. However, the ram effect cannot induce ovulation in cyclic ewes, so is considered ineffective for estrus synchronisation during the breeding season. Conversely,
0093-691X/$ – see front matter. Crown Copyright # 2007 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.03.023
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
there is some evidence that cyclic ewes can respond to stimuli from the ram during the breeding season. For example, during the follicular phase, the continuous presence of rams advances the LH surge [5], accelerates the onset of estrus, and reduces the duration of sexual receptivity [6–11]. During the luteal phase, progesterone produced by the CL strongly inhibits pulsatile LH secretion [12], but ram introduction elicits an increase in pulsatile LH secretion in ovariectomised ewes treated with progesterone during the breeding season [13]. The same applies to seasonally anovulatory ewes undergoing progesterone treatment [14]. More recently, Evans et al. [15] found that ram introduction towards the end of a progestagen synchronisation protocol advanced the LH surge and the onset of estrus after withdrawal of the progestagen. Furthermore, as the rams were removed at progestagen withdrawal, this observation infers a residual effect of rams on the timing of subsequent endocrine events. The apparent ability of ewes to respond to the ram, despite the presence of progesterone, suggests that it may be possible to use the ram to control estrous cycles during the breeding season. We therefore tested whether ram introduction could increase LH pulse frequency in ewes at all stages of the estrous cycle. The responsiveness of ewes to the conventional ram effect is highly dependent on genotype and region of origin [16], so we have done two experiments, one using cyclic Merino ewes, native to Mediterranean regions, and a second using cyclic North of England mule ewes, native to temperate regions. 2. Materials and methods 2.1. Experiment 1 2.1.1. Animals and experimental procedures The experiment was carried out in accordance with the Animals (Scientific Procedures) Act 1986 and was approved by the University of Newcastle Animal Ethics Committee and the UK Home Office. During October (mid-breeding season; Northern Hemisphere), primiparous North of England mule ewes (2 y; Scottish Blackface Bluefaced Leicester; n = 12) that had been previously isolated from ram contact (i.e. more than 500 m away from rams for a minimum of 2 mo) were selected and housed in groups of three in 2 m 1.8 m pens under a photoperiod equivalent to the natural day length (8 h) in an animal house at Cockle Park Research Farm, Newcastle upon Tyne (558130 N). Each pen of
57
ewes was given 2.5 kg hay daily. The rams (n = 1 per three ewes) were adult, sexually experienced, vasectomised, crossbreds (6–7 y; Suffolk-Scottish Blackface Bluefaced Leicester). 2.1.2. Blood sampling Blood was sampled via jugular cannula every 12 min for 6 h before and 6 h after ram introduction (Day 0) and every 2 h from 19 to 43 h after ram introduction (Day 1). This second period was considered best for detecting an LH surge based on observations that, in anovulatory ewes, the ram-induced LH surge occurs 20–40 h after ram introduction [1]. Blood collected for LH was left to clot for a minimum of 16 h at room temperature. Samples were then centrifuged at 2000 g for 15 min and serum was decanted into plastic tubes and stored at 20 8C until analysis. Blood was sampled twice weekly for progesterone for 2 weeks before ram introduction to determine retrospectively the stage of cycle on the day of ram introduction (Day 0). Blood samples were taken daily on Days 3 to 6 after ram introduction, and twice weekly for 3 weeks after ram introduction, to profile estrous cycle dynamics. Blood collected for progesterone was centrifuged immediately at 2000 g for 15 min. Plasma was then decanted into duplicate plastic tubes that were capped, immediately frozen and stored at 20 8C until analysis. 2.1.3. Immunoassay Progesterone was assayed in duplicate samples of plasma using a commercial enzyme linked immunoassay (ELISA) kit (Ridgeway Science Ltd., Gloucester, UK) in accordance with the manufacturer’s instructions, as detailed by Madgwick et al. [17]. The sensitivity of the assay was 0.23 ng/mL. For low (1.85 ng/mL), medium (3.02 ng/mL) and high (7.43 ng/mL) concentration samples, mean intra-assay coefficients of variation were 7.6, 11.4, and 2.7%, and inter-assay coefficients of variation were 10.5, 7.4, and 8.0%. Serum LH concentrations were determined using a previously validated double-antibody radioimmunoassay [18]. The sensitivity of the assay was 0.1 ng/mL. Low (0.28 ng/mL), medium (1.53 ng/mL) and high (3.47 ng/mL) concentration samples were used to estimate mean intra-assay coefficients of variation (l7.1, 16.2, and 7.8%) and mean inter-assay coefficients of variation (8.2, 8.4, and 18.6%). 2.1.4. Data analysis The progesterone profile for each ewe was used to determine the stage of the estrous cycle at the time the
58
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
rams were introduced (Day 0). All ewes were cyclic and were distributed among early luteal (n = 3), mid-luteal (n = 1), late luteal (n = 6), follicular phase (n = 1), late follicular phase-post LH surge (n = 1). Statistical analysis was thus only possible for the early and late luteal phase data. A sample from the beginning and the end of the serial sampling period for each ewe were also analysed to determine progesterone concentrations before and after ram introduction. Estrous cycle length was calculated from the progesterone profiles as the number of days between the nadir points of two successive estrous cycles. For the 12-h blood-sampling period, LH pulses were detected using the Munro algorithm, a modified version of the Pulsar algorithm [19]. The basal concentration of LH was calculated as the mean of the lowest points during the period of observation, as described elsewhere [14]. Data for LH concentration, LH pulse frequency, LH pulse amplitude, basal LH concentrations, before and after ram introduction, were subjected to repeated measures ANOVA in Genstat 5 for Windows (Second Edition, Lawes Agricultural Trust, Rothamsted Experimental Station, UK) to assess the effect of stage of estrous cycle, time relative to ram introduction, and any interactions. Progesterone concentrations at the beginning and end of the frequent sampling period were analysed by repeated measures ANOVA (Genstat 5) to assess any change in progesterone over that period. When a significant effect of ram introduction was detected, data before and after ram introduction were compared by a paired t-test (Minitab, 13.1, Minitab Ltd., Coventry, UK). Log 10 transformation before analysis was used to overcome skewness in the data for progesterone concentrations, basal LH concentrations, and mean LH concentrations. The onset of the LH surge was defined as the sample where the concentrations of LH exceeded the mean of the previous baseline by three standard deviations and were maintained above this level for at least 2 h, as described elsewhere [20]. The end of the surge was defined as the sample where the concentrations of LH returned to this threshold. Ewes detected with an LH surge within the sampling period were recorded for each stage of the estrous cycle. Only ewes classified as in their follicular phase or late luteal phase at the time of ram introduction had an LH surge during the bloodsampling period (follicular phase, 1/1; luteal phase, 4/ 6). The numbers of ewes detected with an LH surge were compared using the Chi-square test (Minitab 13.1).
2.2. Experiment 2 2.2.1. Experimental animals The experiment was carried out in accordance with the Australian code of practice for the care and use of animals for scientific purposes (Seventh Edition, 2004) and was approved by the University of Western Australia Animal Ethics Committee (RA05/100/483). During January (early breeding season; Southern Hemisphere) 32 adult (6–7 y), multiparous Merino ewes that had been previously isolated from ram contact (i.e. more than 500 m away from rams for at minimum of 2 mo) were allocated to one of four groups: early luteal (n = 8), mid-luteal (n = 8), late luteal (n = 8) and follicular phase (n = 8), with each group balanced for age (6–7 y) and live weight (56 5.2 kg). To ensure sufficient replicates for each stage of the cycle, the ewes were synchronised using intravaginal progesterone pessaries (CIDRS; Pacific Vet, Cheltenham, Victoria, Australia) with pessary insertion and withdrawal offset to produce the four treatment groups simultaneously. The stage of the cycle was determined on the basis that ewes came into estrus 2 d after CIDR removal with extra days added to produce each stage: early luteal (+5 d), mid-luteal (+9 d) and late luteal (+13 d). The ewes in the follicular phase group were left for +12 d and treated with prostaglandin F2a (250 mg im Estromil; Troy Laboratories Ltd., Smithfield, New South Wales, Australia) on Day 1 to ensure that their follicular phase coincided with ram introduction (Day 0). Two weeks before the experiment, ewes were moved into an animal facility at the University of Western Australia (318580 S). They were maintained indoors in individual pens under a photoperiod equivalent to the natural day length (13 h) and received a daily ration of 150 g lupin grain, 750 g rough-cut chaff and 25 g minerals. One day before the rams were introduced, each group of ewes was transferred to a group pen in the experimental room to allow acclimatisation to the new environment. Two adult (7 y), sexually experienced, Merino rams were introduced to each group of eight ewes on the day of ram introduction. 2.2.2. Blood sampling To confirm the stage of the estrous cycle on the day of ram introduction, blood was sampled daily from Day 5 to Day 0 in early luteal phase ewes, Day 8 to Day 0 in mid-luteal phase ewes, and Day 12 to Day 0 in late luteal phase ewes. For follicular phase ewes, a single sample was taken on Day 0. To study the LH
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
responses, blood was sampled every 12 min for 4 h before and 2 h after ram introduction for early, midand late luteal phase ewes. For the follicular phase ewes, the sampling frequency was increased to every 5 min to allow for the high endogenous LH pulse frequency typical of this stage of the cycle. All samples were centrifuged at 2000 g for 15 min and plasma was decanted into duplicate plastic tubes that were capped, immediately frozen and stored at 20 8C until analysis. 2.2.3. Immunoassay Plasma LH was measured in duplicate by a doubleantibody radioimmunoassay [21] using ovine LH (NIDDK-oLH-1-4 (AFP-8614B)) for iodination and standards that had been kindly supplied by A. Parlow, National Hormone and Pituitary Program, NIDDK, CA, USA. The sensitivity of the assay was 0.06 ng/mL. Quality control samples (0.63, 1.09, and 1.90 ng/mL) were used to estimate intra-assay coefficients of variation (8.6, 9.9, and 3.8%) and inter-assay coefficients of variation (10.1, 6.7, and 5.2%). Plasma progesterone was measured in duplicate using an active progesterone radioimmunoassay kit (Diagnostic Systems Laboratories Inc., Webster, TX, USA) as described elsewhere [22]. The sensitivity of the assay was 0.1 ng/mL. For low (0.77 ng/mL) and high (9.06 ng/mL) plasma samples, intra-assay coefficients of variation were 4.1 and 3.2%, and inter-assay coefficients of variation were 3.9 and 4.4%. 2.2.4. Data analysis The LH pulses were detected and basal LH concentrations were calculated as outlined for Experiment 1. Data for LH pulse frequency, LH pulse amplitude, mean LH and basal LH concentrations before and after ram introduction were subjected to repeated measures ANOVA in Genstat 5 for Windows (Second Edition, Lawes Agricultural Trust, Rothamsted Experimental Station, UK) to assess the effect of stage of estrous cycle, time relative to ram introduction, and any interactions. Progesterone concentrations at the beginning and end of the frequent sampling period were analysed by repeated measures ANOVA (Genstat 5) to detect changes in progesterone over that period. Where a significant effect of ram introduction was detected, data before and after ram introduction were compared by paired t-test (Genstat 5). Log 10 transformation before analysis was used to overcome skewness in the data for progesterone concentrations, basal LH concentrations and mean LH concentrations.
59
3. Results 3.1. Experiment 1 The ANOVA detected significant effects of time of ram introduction on LH pulse frequency, mean and basal concentrations of LH (at least P < 0.05) but no effect on LH pulse amplitude (P > 0.1). There was no effect of stage of the luteal phase on any characteristics of the LH response (P > 0.1) and no interaction between stage of the luteal phase and time of ram introduction (P > 0.1). All early luteal phase ewes were on Day 2 of the estrous cycle at the time of ram introduction (Fig. 1c). There was an increase in basal and mean concentrations of LH in response to ram introduction (Fig. 1a; P < 0.05) and an increase in LH pulse frequency (Table 1; P < 0.05). However, there was no effect on pulse amplitude (Table 1; P > 0.1) and no LH surges were detected. There was no change in progesterone concentrations over the 12-h frequent sampling period (1.27 0.51 ng/mL versus 1.46 0.50 ng/mL; P > 0.1). Among the six late luteal phase ewes, five ewes were on Day 14 and one was on Day 17 of the estrous cycle at the time of ram introduction. Fig. 1b illustrates the increase in basal and mean concentrations of LH in Table 1 Mean ( S.E.M.) characteristics of the endocrine variables before and after ram introduction to North of England mule ewes at different stages of the estrous cycle Early luteal
Late luteal
No. of ewes
3
6
Progesterone concentration on day of ram introduction (ng/mL)
1.27 0.51
2.47 0.36
LH concentration (ng/mL) Before rams After rams
0.28 0.10 1.13 0.42*
0.31 0.15 0.83 0.20**
LH pulses per hour Before rams After rams
0.28 0.11 0.56 0.11*
0.20 0.07 0.61 0.13*
LH pulse amplitude (ng/mL) Before rams After rams
0.53 0.05 0.46 0.20
0.69 0.43 0.61 0.18
Basal LH concentration (ng/mL) Before rams After rams
0.17 0.02 0.95 0.37*
0.10 0.03 0.56 0.16*
Time from ram introduction to the first pulse (min)
48.0 20.8
36.0 7.59
Values differ within treatment: *P < 0.05; **P < 0.01.
60
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
Fig. 1. Mean (S.E.M.) LH concentrations before and after ram introduction to North of England mule ewes in their early (EL; a) and late (LL; b) luteal phase. (c) and (d) illustrate the mean progesterone concentrations (S.E.M.) on the day of ram introduction of EL and LL ewes. The solid black line in (c) and (d) represents the cycle during which the rams were introduced. The cycle prior to ram introduction was present only in the EL ewes (c) and is represented by a solid grey line. The cycle after ram introduction was present only in the LL ewes and is represented by the dashed black line (d). The arrow indicates the time or day of ram introduction.
response to ram introduction (Table 1; at least P < 0.05). Ram introduction increased LH pulse frequency (P < 0.05), but had no effect on pulse amplitude (Table 1; P > 0.1). An LH surge was detected in four out of six ewes with an average latency of 34.3 4.8 h after ram introduction (excluding one ewe where the latency could not be calculated because the surge began before the sampling period). The concentration of progesterone declined during the 12-h frequent sampling period (2.47 0.36 ng/mL versus 1.65 0.31 ng/mL; P < 0.05). In late luteal phase ewes, the estrous cycle following ram introduction tended to be shorter in length than the cycle during which rams were introduced (17.2 0.65 d versus 15.8 0.54 d; Fig. 1d; P 0.1). A similar analysis was not possible with ewes in their early luteal phase, due to the lack of a nadir point for the cycles before and after ram introduction (Fig. 1c). The follicular, late follicular (post LH surge) and mid-luteal phases were each represented by only one
ewe, so individual LH profiles are presented in Fig. 2. These profiles indicate that the rams induced an LH response at each of these stages. 3.2. Experiment 2 The stage of the estrous cycle on the day of ram introduction was confirmed by the mean progesterone profiles for each group of ewes (Fig. 3). The ANOVA detected significant effects of the stage of estrous cycle and time of ram introduction on LH pulse frequency and mean concentrations of LH (Table 2; at least P < 0.05). There was no effect of stage of cycle or time of ram introduction on LH pulse amplitude (Table 2; P > 0.1). There was an interaction between the stage of estrous cycle and time of ram introduction on mean (Table 2; P < 0.001) and basal concentrations of LH (Table 2; P < 0.05) but not on LH pulse frequency or LH pulse amplitude (Table 2; P > 0.1).
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
61
Fig. 2. Individual profiles of North of England mule ewes in their follicular (a), late follicular (post LH surge; b) and mid-luteal phase (c). The arrow indicates the time of ram introduction.
Specifically, the introduction of rams induced an increase in LH pulse frequency at all stages of the estrous cycle (Table 2; at least P < 0.01). Fig. 4 shows an increase in mean concentrations of LH in ewes in the early, mid- and late luteal phase at the time of ram introduction (Table 2; P < 0.05). This response was not observed in ewes in the follicular phase (Table 2; P > 0.1). Ewes in the early and mid-luteal phase also had an increase in basal concentrations of LH (Table 2;
P < 0.05), but this was not detected in ewes in the late luteal or follicular phases (Table 2; P > 0.1). There was no effect of ram introduction on LH pulse amplitude at any stage of the cycle (Table 2; P > 0.1) and no difference in the delay to the first pulse among the different stages of the cycle (Table 2; P > 0.1). Ewes in the early luteal phase had an increase in progesterone concentrations between the beginning and end of the frequent sampling period (0.98 0.12 ng/mL
62
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
Fig. 3. Profiles of mean (S.E.M.) concentration of progesterone before and on the day of ram introduction (Day 0; arrow) in Merino ewes in their follicular (square; n = 8), early luteal (dash; n = 8), mid-luteal (cross; n = 8) or late luteal phase (diamond; n = 8).
versus 1.20 0.13 ng/mL; P < 0.01). There was no change (P > 0.1) in progesterone concentrations over the 6-h sampling period in ewes in their follicular (0.38 0.09 ng/mL versus 0.31 0.07 ng/mL), midluteal (2.31 0.26 ng/mL versus 3.05 0.49 ng/mL) or late luteal phases (2.38 0.07 ng/mL versus 2.69 0.23 ng/mL).
and luteal phases of the estrous cycle. The response to ram introduction by ewes in their follicular phase was likely to be the driving force behind the advanced LH surge and timing of estrus reported in previous studies [5,9–11]. The robust LH response during the luteal phase, when progesterone concentrations were high, concurred with observations in ewes introduced to rams whilst implanted with progesterone, treated with an artificial progestagen or in the early stages of pregnancy [13–15,23]. Thus, the ability of rams to stimulate the GnRH pulse generator was not blocked by the inhibitory effects of progesterone. It is risky to compare the responsiveness of the two genotypes used in this study, as the experiments were
4. Discussion Cyclic ewes of genotypes that are native to both temperate and Mediterranean regions responded to ram introduction with an increase in LH pulse frequency. This response was evident in ewes in both the follicular
Table 2 Mean (S.E.M.) characteristics of the endocrine variables before and after ram introduction to Merino ewes at different stages of the estrous cycle Follicular
Early luteal
Mid-luteal
Late luteal
No. of ewes
8
8
8
8
Progesterone concentration on day of ram introduction (ng/mL)
0.38 0.09
1.24 0.22
2.31 0.26
2.38 0.07
LH concentration (ng/mL) Before rams After rams
0.32 0.04a 0.38 0.05a
0.28 0.05ab 0.59 0.11*b
0.31 0.09ab 0.65 0.11*b
0.25 0.01b 0.36 0.04*a
LH pulses per hour Before rams After rams
0.50 0.08a 1.36 0.24**
0.21 0.06b 0.88 0.13***
0.29 0.08ab 1.19 0.09**
0.17 0.06b 1.00 0.15***
LH pulse amplitude (ng/mL) Before rams After rams
0.24 0.05 0.24 0.03
0.51 0.06 0.54 0.10
0.38 0.08 0.55 0.10
0.34 0.04 0.30 0.03
Basal LH concentration (ng/mL) Before rams After rams
0.26 0.02 0.29 0.03
0.20 0.03 0.37 0.04*
0.21 0.04 0.35 0.07*
0.21 0.01 0.23 0.02
Time from ram introduction to the first pulse (min)
18.3 7.80
24.0 3.93
27.0 4.94
25.5 8.62
Differences within treatments are indicated by asterisks: *P < 0.05; **P < 0.01; ***P < 0.001. Differences between treatments are indicated by different letters (at least P < 0.05).
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
63
Fig. 4. Mean (S.E.M.) LH concentrations before and after ram introduction to Merino ewes in their follicular (a), early luteal (b), mid-luteal (c) and late luteal phase (d). The arrow indicates the time of ram introduction.
run independently and were not designed for this purpose. However, the observation that rams can elicit an LH response in cyclic ewes native to both regions suggested that this ‘breeding season ram effect’ was independent of the interaction between genotype and environment that causes differences in seasonality among breeds. This contrasts with the ‘conventional ram effect’ where the proportion of ewes ovulating in response to ram introduction varies greatly between breeds [1–3,24]. However, it is notable that the Merino ewes in this study had almost 50% more pulses per hour after ram introduction during the early and late luteal phases than the North of England mule ewes, suggesting a genotypic difference in the responsiveness of cyclic ewes to the ram. This awaits resolution by direct comparison within a single experiment. Fig. 5 summarises the characteristics of LH release at each stage of the cycle and outlines the implications of a ram-induced increase in LH secretion. For example, provision of additional LH during the early luteal phase may affect the developing CL because LH is critical for
progesterone production from the small luteal cells [25]. Changes in progesterone dynamics during the early to mid-luteal phase affect the subsequent timing of the LH surge [26], estrous cycle length [27] and the number of follicle waves before ovulation [28]. During the late luteal phase, there is evidence suggesting that LH can be luteolytic rather than luteotrophic [29]. For example, treatment of ovine endometrium with LH on Day 15 of the estrous cycle increases PGF2/ concentrations [30] and thus may affect the timing of luteolysis. Whether the LH response to rams is of sufficient magnitude to affect the timing of luteolysis is currently unknown and may be contentious as Yildiz et al. [31], found no effect of ram introduction at the time of prostaglandin injection on the subsequent timing of the LH surge. A stimulatory effect of rams on LH secretion during the follicular phase is not always observed [32,33], but an increase in LH pulse frequency in follicular phase ewes, as we have observed, is likely to be the driving force behind the advanced timing of the LH surge, [5] onset and duration of estrus [7,9–11] in ram-exposed ewes.
64
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
Fig. 5. Working hypothesis regarding the characteristics and implications of ram introduction to cyclic ewes during the breeding season.
Finally, as LH pulses play a critical role in follicle development [34], a ram-induced change in LH pulse frequency at any stage of the cycle may have subsidiary effects on follicle wave dynamics and the characteristics of the ovulatory follicle. Thus, the ‘working hypothesis’ outlined in Fig. 5 may explain how the introduction of
males can affect the distribution of estrus in cyclic sheep [36], goats [37] and antelope [38]. In conclusion, ram introduction stimulated an increase in pulsatile LH secretion in cyclic ewes of genotypes native to both Mediterranean and temperate regions. Furthermore, this response was not inhibited by
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
elevated progesterone during the luteal phase. The physiological relevance of the resultant change in the endocrine milieu requires further investigation, but alterations in cycle length and follicular dynamics are possible. Since the LH response is synchronised among females, further investigation may help to develop a strategy for using the ram effect to control estrous cycles during the breeding season. Acknowledgements We thank the Australian Research Council’s Discovery funding scheme (University of Western Australia; Project number DP0558952) and the Yorkshire Agricultural Society (University of Newcastle) for supporting this work. We also thank D. Routledge, A. Fogerty, J. Wightman (University of Newcastle), B. Griffin, and M. Gourlay (University of Western Australia) for their assistance in the care and management of the animals, and C. Bulman, M. Hearn, S. Madgwick, S. Edwards, H. Edge (University of Newcastle), and all the volunteers at the University of Western Australia for their assistance in the data collection. Furthermore, we thank N. Hynes (University College Dublin) and M. Blackberry (University of Western Australia) for their assistance with the LH assays. References [1] Martin GB, Oldham CM, Cognie Y, Pearce DT. The physiological response of anovulatory ewes to the introduction of rams— a review. Live Prod Sci 1986;15:219–47. [2] Rosa HJ, Bryant MJ. The ram effect as a way of modifying the reproductive activity in the ewe: a review. Small Rumin Res 2002;45:1–16. [3] Ungerfeld R, Forsberg M, Rubianes E. Overview of the response of anoestrous ewes to the ram effect. Reprod Fertil Dev 2004;16:479–90. [4] Martin GB, Milton JT, Davidson RH, Banchero Hunzicker GE, Lindsay DR, Blache D. Natural methods for increasing reproductive efficiency in small ruminants. Anim Reprod Sci 2004;82–83:231–45. [5] Lindsay DR, Cognie Y, Pelletier J, Signoret JP. Influence of the presence of rams on the timing of ovulation and discharge of LH in ewes. Physiol Behav 1975;15:423–6. [6] Fletcher IC, Lindsay DR. Effect of rams on the duration of oestrous behaviour in ewes. J Reprod Fertil 1971;25:253–9. [7] Maxwell WMC. Artficial insemination of ewes with frozen, thawed semen at a synchronised oestrus. 1. Effect of time of onset of oestrus, ovulation and insemination on fertility. Anim Reprod Sci 1986;10:301–8. [8] Parsons SD, Hunter GL. Effect of the ram on duration of oestrus in the ewe. J Reprod Fertil 1967;1967:61–70. [9] Romano JE, Christians CJ, Crabo BG. Continuous presence of rams hastens the onset of estrus in ewes synchronized during the breeding season. Appl Anim Behav Sci 2000;66:65–70.
65
[10] Romano JE, Fernandez Abella D, Villegas N. A note on the effect of continuous ram presence on estrus onset, estrus duration and ovulation time in estrus synchronized ewes. Appl Anim Behav Sci 2001;73:193–8. [11] Ungerfeld R, Rubianes E. Estrus response to the ram effect in Corriedale ewes primed with medroxyprogesterone during the breeding season. Small Rumin Res 1999;32:89–91. [12] Baird DT. Pulsatile secretion of LH and ovarian estradiol during the follicular phase of the sheep estrous cycle. Biol Reprod 1978;18:359–64. [13] Pearce DT, Oldham CM. ‘Ram effect’ in the breeding season. Proc Aust Soc Reprod Biol 1983;15:49. [14] Martin GB, Scaramuzzi RJ, Oldham CM, Lindsay DR. Effects of progesterone on the responses of Merino ewes to the introduction of rams during anoestrus. Aust J Biol Sci 1983;36:369–78. [15] Evans AC, Duffy P, Crosby TF, Hawken PA, Boland MP, Beard AP. Effect of ram exposure at the end of progestagen treatment on estrus synchronisation and fertility during the breeding season in ewes. Anim Reprod Sci 2004;84:349–58. [16] Martin GB, Scaramuzzi RJ. The induction of oestrus and ovulation in seasonally anovular ewes by exposure to rams. J Steroid Biochem 1983;19:869–75. [17] Madgwick S, Evans AC, Beard AP. Treating heifers with GnRH from 4 to 8 weeks of age advanced growth and the age at puberty. Theriogenology 2005;63:2323–33. [18] Cooke DJ, Crowe MA, Roche JF. Circulating FSH isoform patterns during recurrent increases in FSH throughout the oestrous cycle of heifers. J Reprod Fertil 1997;110:339–45. [19] Merriam GR, Wachter KW. Algorithms for the study of episodic hormone secretion. Am J Physiol 1982;243:E310–8. [20] Caraty A, Delaleu B, Chesneau D, Fabre-nys C. Sequential role of E2 and GnRH for the expression of estrous behavior in ewes. Endocrinology 2002;143:139–45. [21] Martin GB, Oldham CM, Lindsay DR. Increased plasma LH levels in seasonally anovular Merino ewes following the introduction of rams. Anim Reprod Sci 1980;3. [22] Gray CA, Bartol FF, Taylor KM, Wiley AA, Ramsey WS, Ott TL, et al. Ovine uterine gland knock-out model: effects of gland ablation on the estrous cycle. Biol Reprod 2000;62:448–56. [23] Al-Gubory KH. Effects of the presence of rams during pregnancy on lambing performance in ewes. Anim Reprod Sci 1998;52:205–11. [24] Walkden-Brown SW, Martin GB, Restall BJ. Role of malefemale interactions in regulating reproduction in sheep and goats. J Reprod Fertil Suppl 1999;52. [25] Niswender GD, Juengel JL, McGuire WJ, Belfiore CJ, Wiltbank MC. Luteal function: the estrous cycle and early pregnancy. Biol Reprod 1994;50:239–47. [26] Skinner DC, Harris TG, Evans NP. Duration and amplitude of the luteal phase progesterone increment times the estradiol-induced luteinizing hormone surge in ewes. Biol Reprod 2000;63:1135–42. [27] Nephew KP, McClure KE, Ott TL, Dubois DH, Bazer FW, Pope WF. Relationship between variation in conceptus development and differences in estrous cycle duration in ewes. Biol Reprod 1991;44:536–9. [28] Menchaca A, Rubianes E. Relation between progesterone concentrations during the early luteal phase and follicular dynamics in goats. Theriogenology 2002;57:1411–9. [29] Ziecek AJ, Bodek G, Blitek A, Kaczmarek A, Waclawik N. Nongonadal LH receptors, their involvement in female reproductive function and a new applicable approach. Vet J 2005;169: 75–84.
66
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
[30] Weems YS, Kim L, Humphreys V, Tsuda V, Weems CW. Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E2 (PGE2) and F2alpha (PGF2alpha) and progesterone in vitro. Prostaglandins Other Lipid Mediat 2003;71:55–73. [31] Yildiz S, Saatci M, Uzun M, Guven B. Effects of ram introduction after the second prostaglandin F2 alpha injection on day 11 on the LH surge characteristics in fat-tailed ewes. Reprod Domest Anim 2003;38:54–7. [32] Yildiz S, Metehan U, Mehmet K. Effects of rams and luteal or follicular phase ewes on preovulatory LH surge characteristics in ewes. Turk J Vet Anim Sci 2004;28:669–73. [33] Ungerfeld R, Carbajal B, Rubianes E, Forsberg M. Endocrine and ovarian changes in response to the ram effect in medrox-
[34]
[36]
[37]
[38]
yprogesterone acetate-primed Corriedale ewes during the breeding and nonbreeding season. Acta Vet Scand 2005;46:33–44. Scaramuzzi RJ, Adams NR, Baird DT, Campbell BK, Downing JA, Findlay JK, et al. A model for follicle selection and the determination of ovulation rate in the ewe. Reprod Fertil Dev 1993;5:459–78. Ngere LO, Dzakuma JM. The effect of sudden introduction of rams on oestrous pattern of tropical ewes. J Agric Sci 1975;84. Chemineau P. Effect on oestrus and ovulation of exposing creole goats to the male at three times of the year. J Reprod Fertil 1983;67:65–72. Skinner DC, Cilliers SD, Skinner JD. Effect of ram introduction on the oestrous cycle of springbok ewes (Antidorcas marsupialis). Reproduction 2002;124:509–13.
The introduction of rams induces an increase in pulsatile LH secretion in cyclic ewes during the breeding season P.A.R. Hawken a,b,*, A.P. Beard b, T. Esmaili a, H. Kadokawa c, A.C.O. Evans d, D. Blache a, G.B. Martin a a School of Animal Biology, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia School of Agriculture, Food and Rural Development, University of Newcastle, Newcastle upon Tyne NE1 7RU, United Kingdom c Department of Veterinary Medicine, Faculty of Agricultural Science, The University of Yamaguchi, Yoshida 1677-1, Yamaguchi 753-8515, Japan d School of Agriculture, Food Science and Veterinary Medicine, Conway Institute for Biomolecular and Biomedical Research, College of Life Science, University College Dublin, Belfield, Dublin 4, Ireland b
Received 5 February 2007; accepted 17 March 2007
Abstract Application of the ram effect during the breeding season has been previously disregarded because the ewe reproductive axis is powerfully inhibited by luteal phase progesterone concentrations. However, anovulatory ewes treated with exogenous progestagens respond to ram introduction with an increase in LH concentrations. We therefore tested whether cyclic ewes would respond to ram introduction with an increase in pulsatile LH secretion at all stages of the estrous cycle. We did two experiments using genotypes native to temperate or Mediterranean regions. In Experiment 1 (UK), 12 randomly cycling, North of England Mule ewes were introduced to rams midway through a frequent blood-sampling regime. Ewes in the early (EL; n = 6) and late luteal (LL; n = 6) phase responded to ram introduction with an increase in LH pulse frequency and mean and basal concentrations of LH (at least P < 0.05). In Experiment 2 (Australia), the cycles of 32 Merino ewes were synchronised using intravaginal progestagen pessaries. Pessary insertion was staggered to produce eight ewes at each stage of the estrous cycle: follicular (F), early luteal (EL), mid-luteal (ML) and late luteal (LL). In all stages of the cycle, ewes responded to ram introduction with an increase in LH pulse frequency (P < 0.01); EL, ML and LL ewes also had an increase in mean LH concentration (P < 0.05). In conclusion, ram introduction to cyclic ewes stimulated an increase in pulsatile LH secretion, independent of ewe genotype or stage of the estrous cycle. Crown Copyright # 2007 Published by Elsevier Inc. All rights reserved. Keywords: Ram effect; Breeding season; Estrous cycle; Ewes; Luteinising hormone
1. Introduction The ram effect is a well-established phenomenon that induces ovulation in anovulatory ewes. In brief, the introduction of rams stimulates an increase in pulsatile
* Corresponding author at: School of Animal Biology M085, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Tel.: +61 8 6488 3588; fax: +61 8 6488 1040. E-mail address: [email protected] (P.A.R. Hawken).
secretion of LH within minutes and, in some breeds, this is sufficient to induce a pre-ovulatory LH surge, ovulation and, subsequently, estrus and conception [1–3]. One of the subsidiary benefits is that the response to the ram effect is synchronous among the flock so it can be used as a non-pharmacological alternative to conventional methods of estrus synchronisation, an important issue in today’s consumer-driven climate [4]. However, the ram effect cannot induce ovulation in cyclic ewes, so is considered ineffective for estrus synchronisation during the breeding season. Conversely,
0093-691X/$ – see front matter. Crown Copyright # 2007 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.03.023
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
there is some evidence that cyclic ewes can respond to stimuli from the ram during the breeding season. For example, during the follicular phase, the continuous presence of rams advances the LH surge [5], accelerates the onset of estrus, and reduces the duration of sexual receptivity [6–11]. During the luteal phase, progesterone produced by the CL strongly inhibits pulsatile LH secretion [12], but ram introduction elicits an increase in pulsatile LH secretion in ovariectomised ewes treated with progesterone during the breeding season [13]. The same applies to seasonally anovulatory ewes undergoing progesterone treatment [14]. More recently, Evans et al. [15] found that ram introduction towards the end of a progestagen synchronisation protocol advanced the LH surge and the onset of estrus after withdrawal of the progestagen. Furthermore, as the rams were removed at progestagen withdrawal, this observation infers a residual effect of rams on the timing of subsequent endocrine events. The apparent ability of ewes to respond to the ram, despite the presence of progesterone, suggests that it may be possible to use the ram to control estrous cycles during the breeding season. We therefore tested whether ram introduction could increase LH pulse frequency in ewes at all stages of the estrous cycle. The responsiveness of ewes to the conventional ram effect is highly dependent on genotype and region of origin [16], so we have done two experiments, one using cyclic Merino ewes, native to Mediterranean regions, and a second using cyclic North of England mule ewes, native to temperate regions. 2. Materials and methods 2.1. Experiment 1 2.1.1. Animals and experimental procedures The experiment was carried out in accordance with the Animals (Scientific Procedures) Act 1986 and was approved by the University of Newcastle Animal Ethics Committee and the UK Home Office. During October (mid-breeding season; Northern Hemisphere), primiparous North of England mule ewes (2 y; Scottish Blackface Bluefaced Leicester; n = 12) that had been previously isolated from ram contact (i.e. more than 500 m away from rams for a minimum of 2 mo) were selected and housed in groups of three in 2 m 1.8 m pens under a photoperiod equivalent to the natural day length (8 h) in an animal house at Cockle Park Research Farm, Newcastle upon Tyne (558130 N). Each pen of
57
ewes was given 2.5 kg hay daily. The rams (n = 1 per three ewes) were adult, sexually experienced, vasectomised, crossbreds (6–7 y; Suffolk-Scottish Blackface Bluefaced Leicester). 2.1.2. Blood sampling Blood was sampled via jugular cannula every 12 min for 6 h before and 6 h after ram introduction (Day 0) and every 2 h from 19 to 43 h after ram introduction (Day 1). This second period was considered best for detecting an LH surge based on observations that, in anovulatory ewes, the ram-induced LH surge occurs 20–40 h after ram introduction [1]. Blood collected for LH was left to clot for a minimum of 16 h at room temperature. Samples were then centrifuged at 2000 g for 15 min and serum was decanted into plastic tubes and stored at 20 8C until analysis. Blood was sampled twice weekly for progesterone for 2 weeks before ram introduction to determine retrospectively the stage of cycle on the day of ram introduction (Day 0). Blood samples were taken daily on Days 3 to 6 after ram introduction, and twice weekly for 3 weeks after ram introduction, to profile estrous cycle dynamics. Blood collected for progesterone was centrifuged immediately at 2000 g for 15 min. Plasma was then decanted into duplicate plastic tubes that were capped, immediately frozen and stored at 20 8C until analysis. 2.1.3. Immunoassay Progesterone was assayed in duplicate samples of plasma using a commercial enzyme linked immunoassay (ELISA) kit (Ridgeway Science Ltd., Gloucester, UK) in accordance with the manufacturer’s instructions, as detailed by Madgwick et al. [17]. The sensitivity of the assay was 0.23 ng/mL. For low (1.85 ng/mL), medium (3.02 ng/mL) and high (7.43 ng/mL) concentration samples, mean intra-assay coefficients of variation were 7.6, 11.4, and 2.7%, and inter-assay coefficients of variation were 10.5, 7.4, and 8.0%. Serum LH concentrations were determined using a previously validated double-antibody radioimmunoassay [18]. The sensitivity of the assay was 0.1 ng/mL. Low (0.28 ng/mL), medium (1.53 ng/mL) and high (3.47 ng/mL) concentration samples were used to estimate mean intra-assay coefficients of variation (l7.1, 16.2, and 7.8%) and mean inter-assay coefficients of variation (8.2, 8.4, and 18.6%). 2.1.4. Data analysis The progesterone profile for each ewe was used to determine the stage of the estrous cycle at the time the
58
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
rams were introduced (Day 0). All ewes were cyclic and were distributed among early luteal (n = 3), mid-luteal (n = 1), late luteal (n = 6), follicular phase (n = 1), late follicular phase-post LH surge (n = 1). Statistical analysis was thus only possible for the early and late luteal phase data. A sample from the beginning and the end of the serial sampling period for each ewe were also analysed to determine progesterone concentrations before and after ram introduction. Estrous cycle length was calculated from the progesterone profiles as the number of days between the nadir points of two successive estrous cycles. For the 12-h blood-sampling period, LH pulses were detected using the Munro algorithm, a modified version of the Pulsar algorithm [19]. The basal concentration of LH was calculated as the mean of the lowest points during the period of observation, as described elsewhere [14]. Data for LH concentration, LH pulse frequency, LH pulse amplitude, basal LH concentrations, before and after ram introduction, were subjected to repeated measures ANOVA in Genstat 5 for Windows (Second Edition, Lawes Agricultural Trust, Rothamsted Experimental Station, UK) to assess the effect of stage of estrous cycle, time relative to ram introduction, and any interactions. Progesterone concentrations at the beginning and end of the frequent sampling period were analysed by repeated measures ANOVA (Genstat 5) to assess any change in progesterone over that period. When a significant effect of ram introduction was detected, data before and after ram introduction were compared by a paired t-test (Minitab, 13.1, Minitab Ltd., Coventry, UK). Log 10 transformation before analysis was used to overcome skewness in the data for progesterone concentrations, basal LH concentrations, and mean LH concentrations. The onset of the LH surge was defined as the sample where the concentrations of LH exceeded the mean of the previous baseline by three standard deviations and were maintained above this level for at least 2 h, as described elsewhere [20]. The end of the surge was defined as the sample where the concentrations of LH returned to this threshold. Ewes detected with an LH surge within the sampling period were recorded for each stage of the estrous cycle. Only ewes classified as in their follicular phase or late luteal phase at the time of ram introduction had an LH surge during the bloodsampling period (follicular phase, 1/1; luteal phase, 4/ 6). The numbers of ewes detected with an LH surge were compared using the Chi-square test (Minitab 13.1).
2.2. Experiment 2 2.2.1. Experimental animals The experiment was carried out in accordance with the Australian code of practice for the care and use of animals for scientific purposes (Seventh Edition, 2004) and was approved by the University of Western Australia Animal Ethics Committee (RA05/100/483). During January (early breeding season; Southern Hemisphere) 32 adult (6–7 y), multiparous Merino ewes that had been previously isolated from ram contact (i.e. more than 500 m away from rams for at minimum of 2 mo) were allocated to one of four groups: early luteal (n = 8), mid-luteal (n = 8), late luteal (n = 8) and follicular phase (n = 8), with each group balanced for age (6–7 y) and live weight (56 5.2 kg). To ensure sufficient replicates for each stage of the cycle, the ewes were synchronised using intravaginal progesterone pessaries (CIDRS; Pacific Vet, Cheltenham, Victoria, Australia) with pessary insertion and withdrawal offset to produce the four treatment groups simultaneously. The stage of the cycle was determined on the basis that ewes came into estrus 2 d after CIDR removal with extra days added to produce each stage: early luteal (+5 d), mid-luteal (+9 d) and late luteal (+13 d). The ewes in the follicular phase group were left for +12 d and treated with prostaglandin F2a (250 mg im Estromil; Troy Laboratories Ltd., Smithfield, New South Wales, Australia) on Day 1 to ensure that their follicular phase coincided with ram introduction (Day 0). Two weeks before the experiment, ewes were moved into an animal facility at the University of Western Australia (318580 S). They were maintained indoors in individual pens under a photoperiod equivalent to the natural day length (13 h) and received a daily ration of 150 g lupin grain, 750 g rough-cut chaff and 25 g minerals. One day before the rams were introduced, each group of ewes was transferred to a group pen in the experimental room to allow acclimatisation to the new environment. Two adult (7 y), sexually experienced, Merino rams were introduced to each group of eight ewes on the day of ram introduction. 2.2.2. Blood sampling To confirm the stage of the estrous cycle on the day of ram introduction, blood was sampled daily from Day 5 to Day 0 in early luteal phase ewes, Day 8 to Day 0 in mid-luteal phase ewes, and Day 12 to Day 0 in late luteal phase ewes. For follicular phase ewes, a single sample was taken on Day 0. To study the LH
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
responses, blood was sampled every 12 min for 4 h before and 2 h after ram introduction for early, midand late luteal phase ewes. For the follicular phase ewes, the sampling frequency was increased to every 5 min to allow for the high endogenous LH pulse frequency typical of this stage of the cycle. All samples were centrifuged at 2000 g for 15 min and plasma was decanted into duplicate plastic tubes that were capped, immediately frozen and stored at 20 8C until analysis. 2.2.3. Immunoassay Plasma LH was measured in duplicate by a doubleantibody radioimmunoassay [21] using ovine LH (NIDDK-oLH-1-4 (AFP-8614B)) for iodination and standards that had been kindly supplied by A. Parlow, National Hormone and Pituitary Program, NIDDK, CA, USA. The sensitivity of the assay was 0.06 ng/mL. Quality control samples (0.63, 1.09, and 1.90 ng/mL) were used to estimate intra-assay coefficients of variation (8.6, 9.9, and 3.8%) and inter-assay coefficients of variation (10.1, 6.7, and 5.2%). Plasma progesterone was measured in duplicate using an active progesterone radioimmunoassay kit (Diagnostic Systems Laboratories Inc., Webster, TX, USA) as described elsewhere [22]. The sensitivity of the assay was 0.1 ng/mL. For low (0.77 ng/mL) and high (9.06 ng/mL) plasma samples, intra-assay coefficients of variation were 4.1 and 3.2%, and inter-assay coefficients of variation were 3.9 and 4.4%. 2.2.4. Data analysis The LH pulses were detected and basal LH concentrations were calculated as outlined for Experiment 1. Data for LH pulse frequency, LH pulse amplitude, mean LH and basal LH concentrations before and after ram introduction were subjected to repeated measures ANOVA in Genstat 5 for Windows (Second Edition, Lawes Agricultural Trust, Rothamsted Experimental Station, UK) to assess the effect of stage of estrous cycle, time relative to ram introduction, and any interactions. Progesterone concentrations at the beginning and end of the frequent sampling period were analysed by repeated measures ANOVA (Genstat 5) to detect changes in progesterone over that period. Where a significant effect of ram introduction was detected, data before and after ram introduction were compared by paired t-test (Genstat 5). Log 10 transformation before analysis was used to overcome skewness in the data for progesterone concentrations, basal LH concentrations and mean LH concentrations.
59
3. Results 3.1. Experiment 1 The ANOVA detected significant effects of time of ram introduction on LH pulse frequency, mean and basal concentrations of LH (at least P < 0.05) but no effect on LH pulse amplitude (P > 0.1). There was no effect of stage of the luteal phase on any characteristics of the LH response (P > 0.1) and no interaction between stage of the luteal phase and time of ram introduction (P > 0.1). All early luteal phase ewes were on Day 2 of the estrous cycle at the time of ram introduction (Fig. 1c). There was an increase in basal and mean concentrations of LH in response to ram introduction (Fig. 1a; P < 0.05) and an increase in LH pulse frequency (Table 1; P < 0.05). However, there was no effect on pulse amplitude (Table 1; P > 0.1) and no LH surges were detected. There was no change in progesterone concentrations over the 12-h frequent sampling period (1.27 0.51 ng/mL versus 1.46 0.50 ng/mL; P > 0.1). Among the six late luteal phase ewes, five ewes were on Day 14 and one was on Day 17 of the estrous cycle at the time of ram introduction. Fig. 1b illustrates the increase in basal and mean concentrations of LH in Table 1 Mean ( S.E.M.) characteristics of the endocrine variables before and after ram introduction to North of England mule ewes at different stages of the estrous cycle Early luteal
Late luteal
No. of ewes
3
6
Progesterone concentration on day of ram introduction (ng/mL)
1.27 0.51
2.47 0.36
LH concentration (ng/mL) Before rams After rams
0.28 0.10 1.13 0.42*
0.31 0.15 0.83 0.20**
LH pulses per hour Before rams After rams
0.28 0.11 0.56 0.11*
0.20 0.07 0.61 0.13*
LH pulse amplitude (ng/mL) Before rams After rams
0.53 0.05 0.46 0.20
0.69 0.43 0.61 0.18
Basal LH concentration (ng/mL) Before rams After rams
0.17 0.02 0.95 0.37*
0.10 0.03 0.56 0.16*
Time from ram introduction to the first pulse (min)
48.0 20.8
36.0 7.59
Values differ within treatment: *P < 0.05; **P < 0.01.
60
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
Fig. 1. Mean (S.E.M.) LH concentrations before and after ram introduction to North of England mule ewes in their early (EL; a) and late (LL; b) luteal phase. (c) and (d) illustrate the mean progesterone concentrations (S.E.M.) on the day of ram introduction of EL and LL ewes. The solid black line in (c) and (d) represents the cycle during which the rams were introduced. The cycle prior to ram introduction was present only in the EL ewes (c) and is represented by a solid grey line. The cycle after ram introduction was present only in the LL ewes and is represented by the dashed black line (d). The arrow indicates the time or day of ram introduction.
response to ram introduction (Table 1; at least P < 0.05). Ram introduction increased LH pulse frequency (P < 0.05), but had no effect on pulse amplitude (Table 1; P > 0.1). An LH surge was detected in four out of six ewes with an average latency of 34.3 4.8 h after ram introduction (excluding one ewe where the latency could not be calculated because the surge began before the sampling period). The concentration of progesterone declined during the 12-h frequent sampling period (2.47 0.36 ng/mL versus 1.65 0.31 ng/mL; P < 0.05). In late luteal phase ewes, the estrous cycle following ram introduction tended to be shorter in length than the cycle during which rams were introduced (17.2 0.65 d versus 15.8 0.54 d; Fig. 1d; P 0.1). A similar analysis was not possible with ewes in their early luteal phase, due to the lack of a nadir point for the cycles before and after ram introduction (Fig. 1c). The follicular, late follicular (post LH surge) and mid-luteal phases were each represented by only one
ewe, so individual LH profiles are presented in Fig. 2. These profiles indicate that the rams induced an LH response at each of these stages. 3.2. Experiment 2 The stage of the estrous cycle on the day of ram introduction was confirmed by the mean progesterone profiles for each group of ewes (Fig. 3). The ANOVA detected significant effects of the stage of estrous cycle and time of ram introduction on LH pulse frequency and mean concentrations of LH (Table 2; at least P < 0.05). There was no effect of stage of cycle or time of ram introduction on LH pulse amplitude (Table 2; P > 0.1). There was an interaction between the stage of estrous cycle and time of ram introduction on mean (Table 2; P < 0.001) and basal concentrations of LH (Table 2; P < 0.05) but not on LH pulse frequency or LH pulse amplitude (Table 2; P > 0.1).
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
61
Fig. 2. Individual profiles of North of England mule ewes in their follicular (a), late follicular (post LH surge; b) and mid-luteal phase (c). The arrow indicates the time of ram introduction.
Specifically, the introduction of rams induced an increase in LH pulse frequency at all stages of the estrous cycle (Table 2; at least P < 0.01). Fig. 4 shows an increase in mean concentrations of LH in ewes in the early, mid- and late luteal phase at the time of ram introduction (Table 2; P < 0.05). This response was not observed in ewes in the follicular phase (Table 2; P > 0.1). Ewes in the early and mid-luteal phase also had an increase in basal concentrations of LH (Table 2;
P < 0.05), but this was not detected in ewes in the late luteal or follicular phases (Table 2; P > 0.1). There was no effect of ram introduction on LH pulse amplitude at any stage of the cycle (Table 2; P > 0.1) and no difference in the delay to the first pulse among the different stages of the cycle (Table 2; P > 0.1). Ewes in the early luteal phase had an increase in progesterone concentrations between the beginning and end of the frequent sampling period (0.98 0.12 ng/mL
62
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
Fig. 3. Profiles of mean (S.E.M.) concentration of progesterone before and on the day of ram introduction (Day 0; arrow) in Merino ewes in their follicular (square; n = 8), early luteal (dash; n = 8), mid-luteal (cross; n = 8) or late luteal phase (diamond; n = 8).
versus 1.20 0.13 ng/mL; P < 0.01). There was no change (P > 0.1) in progesterone concentrations over the 6-h sampling period in ewes in their follicular (0.38 0.09 ng/mL versus 0.31 0.07 ng/mL), midluteal (2.31 0.26 ng/mL versus 3.05 0.49 ng/mL) or late luteal phases (2.38 0.07 ng/mL versus 2.69 0.23 ng/mL).
and luteal phases of the estrous cycle. The response to ram introduction by ewes in their follicular phase was likely to be the driving force behind the advanced LH surge and timing of estrus reported in previous studies [5,9–11]. The robust LH response during the luteal phase, when progesterone concentrations were high, concurred with observations in ewes introduced to rams whilst implanted with progesterone, treated with an artificial progestagen or in the early stages of pregnancy [13–15,23]. Thus, the ability of rams to stimulate the GnRH pulse generator was not blocked by the inhibitory effects of progesterone. It is risky to compare the responsiveness of the two genotypes used in this study, as the experiments were
4. Discussion Cyclic ewes of genotypes that are native to both temperate and Mediterranean regions responded to ram introduction with an increase in LH pulse frequency. This response was evident in ewes in both the follicular
Table 2 Mean (S.E.M.) characteristics of the endocrine variables before and after ram introduction to Merino ewes at different stages of the estrous cycle Follicular
Early luteal
Mid-luteal
Late luteal
No. of ewes
8
8
8
8
Progesterone concentration on day of ram introduction (ng/mL)
0.38 0.09
1.24 0.22
2.31 0.26
2.38 0.07
LH concentration (ng/mL) Before rams After rams
0.32 0.04a 0.38 0.05a
0.28 0.05ab 0.59 0.11*b
0.31 0.09ab 0.65 0.11*b
0.25 0.01b 0.36 0.04*a
LH pulses per hour Before rams After rams
0.50 0.08a 1.36 0.24**
0.21 0.06b 0.88 0.13***
0.29 0.08ab 1.19 0.09**
0.17 0.06b 1.00 0.15***
LH pulse amplitude (ng/mL) Before rams After rams
0.24 0.05 0.24 0.03
0.51 0.06 0.54 0.10
0.38 0.08 0.55 0.10
0.34 0.04 0.30 0.03
Basal LH concentration (ng/mL) Before rams After rams
0.26 0.02 0.29 0.03
0.20 0.03 0.37 0.04*
0.21 0.04 0.35 0.07*
0.21 0.01 0.23 0.02
Time from ram introduction to the first pulse (min)
18.3 7.80
24.0 3.93
27.0 4.94
25.5 8.62
Differences within treatments are indicated by asterisks: *P < 0.05; **P < 0.01; ***P < 0.001. Differences between treatments are indicated by different letters (at least P < 0.05).
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
63
Fig. 4. Mean (S.E.M.) LH concentrations before and after ram introduction to Merino ewes in their follicular (a), early luteal (b), mid-luteal (c) and late luteal phase (d). The arrow indicates the time of ram introduction.
run independently and were not designed for this purpose. However, the observation that rams can elicit an LH response in cyclic ewes native to both regions suggested that this ‘breeding season ram effect’ was independent of the interaction between genotype and environment that causes differences in seasonality among breeds. This contrasts with the ‘conventional ram effect’ where the proportion of ewes ovulating in response to ram introduction varies greatly between breeds [1–3,24]. However, it is notable that the Merino ewes in this study had almost 50% more pulses per hour after ram introduction during the early and late luteal phases than the North of England mule ewes, suggesting a genotypic difference in the responsiveness of cyclic ewes to the ram. This awaits resolution by direct comparison within a single experiment. Fig. 5 summarises the characteristics of LH release at each stage of the cycle and outlines the implications of a ram-induced increase in LH secretion. For example, provision of additional LH during the early luteal phase may affect the developing CL because LH is critical for
progesterone production from the small luteal cells [25]. Changes in progesterone dynamics during the early to mid-luteal phase affect the subsequent timing of the LH surge [26], estrous cycle length [27] and the number of follicle waves before ovulation [28]. During the late luteal phase, there is evidence suggesting that LH can be luteolytic rather than luteotrophic [29]. For example, treatment of ovine endometrium with LH on Day 15 of the estrous cycle increases PGF2/ concentrations [30] and thus may affect the timing of luteolysis. Whether the LH response to rams is of sufficient magnitude to affect the timing of luteolysis is currently unknown and may be contentious as Yildiz et al. [31], found no effect of ram introduction at the time of prostaglandin injection on the subsequent timing of the LH surge. A stimulatory effect of rams on LH secretion during the follicular phase is not always observed [32,33], but an increase in LH pulse frequency in follicular phase ewes, as we have observed, is likely to be the driving force behind the advanced timing of the LH surge, [5] onset and duration of estrus [7,9–11] in ram-exposed ewes.
64
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
Fig. 5. Working hypothesis regarding the characteristics and implications of ram introduction to cyclic ewes during the breeding season.
Finally, as LH pulses play a critical role in follicle development [34], a ram-induced change in LH pulse frequency at any stage of the cycle may have subsidiary effects on follicle wave dynamics and the characteristics of the ovulatory follicle. Thus, the ‘working hypothesis’ outlined in Fig. 5 may explain how the introduction of
males can affect the distribution of estrus in cyclic sheep [36], goats [37] and antelope [38]. In conclusion, ram introduction stimulated an increase in pulsatile LH secretion in cyclic ewes of genotypes native to both Mediterranean and temperate regions. Furthermore, this response was not inhibited by
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
elevated progesterone during the luteal phase. The physiological relevance of the resultant change in the endocrine milieu requires further investigation, but alterations in cycle length and follicular dynamics are possible. Since the LH response is synchronised among females, further investigation may help to develop a strategy for using the ram effect to control estrous cycles during the breeding season. Acknowledgements We thank the Australian Research Council’s Discovery funding scheme (University of Western Australia; Project number DP0558952) and the Yorkshire Agricultural Society (University of Newcastle) for supporting this work. We also thank D. Routledge, A. Fogerty, J. Wightman (University of Newcastle), B. Griffin, and M. Gourlay (University of Western Australia) for their assistance in the care and management of the animals, and C. Bulman, M. Hearn, S. Madgwick, S. Edwards, H. Edge (University of Newcastle), and all the volunteers at the University of Western Australia for their assistance in the data collection. Furthermore, we thank N. Hynes (University College Dublin) and M. Blackberry (University of Western Australia) for their assistance with the LH assays. References [1] Martin GB, Oldham CM, Cognie Y, Pearce DT. The physiological response of anovulatory ewes to the introduction of rams— a review. Live Prod Sci 1986;15:219–47. [2] Rosa HJ, Bryant MJ. The ram effect as a way of modifying the reproductive activity in the ewe: a review. Small Rumin Res 2002;45:1–16. [3] Ungerfeld R, Forsberg M, Rubianes E. Overview of the response of anoestrous ewes to the ram effect. Reprod Fertil Dev 2004;16:479–90. [4] Martin GB, Milton JT, Davidson RH, Banchero Hunzicker GE, Lindsay DR, Blache D. Natural methods for increasing reproductive efficiency in small ruminants. Anim Reprod Sci 2004;82–83:231–45. [5] Lindsay DR, Cognie Y, Pelletier J, Signoret JP. Influence of the presence of rams on the timing of ovulation and discharge of LH in ewes. Physiol Behav 1975;15:423–6. [6] Fletcher IC, Lindsay DR. Effect of rams on the duration of oestrous behaviour in ewes. J Reprod Fertil 1971;25:253–9. [7] Maxwell WMC. Artficial insemination of ewes with frozen, thawed semen at a synchronised oestrus. 1. Effect of time of onset of oestrus, ovulation and insemination on fertility. Anim Reprod Sci 1986;10:301–8. [8] Parsons SD, Hunter GL. Effect of the ram on duration of oestrus in the ewe. J Reprod Fertil 1967;1967:61–70. [9] Romano JE, Christians CJ, Crabo BG. Continuous presence of rams hastens the onset of estrus in ewes synchronized during the breeding season. Appl Anim Behav Sci 2000;66:65–70.
65
[10] Romano JE, Fernandez Abella D, Villegas N. A note on the effect of continuous ram presence on estrus onset, estrus duration and ovulation time in estrus synchronized ewes. Appl Anim Behav Sci 2001;73:193–8. [11] Ungerfeld R, Rubianes E. Estrus response to the ram effect in Corriedale ewes primed with medroxyprogesterone during the breeding season. Small Rumin Res 1999;32:89–91. [12] Baird DT. Pulsatile secretion of LH and ovarian estradiol during the follicular phase of the sheep estrous cycle. Biol Reprod 1978;18:359–64. [13] Pearce DT, Oldham CM. ‘Ram effect’ in the breeding season. Proc Aust Soc Reprod Biol 1983;15:49. [14] Martin GB, Scaramuzzi RJ, Oldham CM, Lindsay DR. Effects of progesterone on the responses of Merino ewes to the introduction of rams during anoestrus. Aust J Biol Sci 1983;36:369–78. [15] Evans AC, Duffy P, Crosby TF, Hawken PA, Boland MP, Beard AP. Effect of ram exposure at the end of progestagen treatment on estrus synchronisation and fertility during the breeding season in ewes. Anim Reprod Sci 2004;84:349–58. [16] Martin GB, Scaramuzzi RJ. The induction of oestrus and ovulation in seasonally anovular ewes by exposure to rams. J Steroid Biochem 1983;19:869–75. [17] Madgwick S, Evans AC, Beard AP. Treating heifers with GnRH from 4 to 8 weeks of age advanced growth and the age at puberty. Theriogenology 2005;63:2323–33. [18] Cooke DJ, Crowe MA, Roche JF. Circulating FSH isoform patterns during recurrent increases in FSH throughout the oestrous cycle of heifers. J Reprod Fertil 1997;110:339–45. [19] Merriam GR, Wachter KW. Algorithms for the study of episodic hormone secretion. Am J Physiol 1982;243:E310–8. [20] Caraty A, Delaleu B, Chesneau D, Fabre-nys C. Sequential role of E2 and GnRH for the expression of estrous behavior in ewes. Endocrinology 2002;143:139–45. [21] Martin GB, Oldham CM, Lindsay DR. Increased plasma LH levels in seasonally anovular Merino ewes following the introduction of rams. Anim Reprod Sci 1980;3. [22] Gray CA, Bartol FF, Taylor KM, Wiley AA, Ramsey WS, Ott TL, et al. Ovine uterine gland knock-out model: effects of gland ablation on the estrous cycle. Biol Reprod 2000;62:448–56. [23] Al-Gubory KH. Effects of the presence of rams during pregnancy on lambing performance in ewes. Anim Reprod Sci 1998;52:205–11. [24] Walkden-Brown SW, Martin GB, Restall BJ. Role of malefemale interactions in regulating reproduction in sheep and goats. J Reprod Fertil Suppl 1999;52. [25] Niswender GD, Juengel JL, McGuire WJ, Belfiore CJ, Wiltbank MC. Luteal function: the estrous cycle and early pregnancy. Biol Reprod 1994;50:239–47. [26] Skinner DC, Harris TG, Evans NP. Duration and amplitude of the luteal phase progesterone increment times the estradiol-induced luteinizing hormone surge in ewes. Biol Reprod 2000;63:1135–42. [27] Nephew KP, McClure KE, Ott TL, Dubois DH, Bazer FW, Pope WF. Relationship between variation in conceptus development and differences in estrous cycle duration in ewes. Biol Reprod 1991;44:536–9. [28] Menchaca A, Rubianes E. Relation between progesterone concentrations during the early luteal phase and follicular dynamics in goats. Theriogenology 2002;57:1411–9. [29] Ziecek AJ, Bodek G, Blitek A, Kaczmarek A, Waclawik N. Nongonadal LH receptors, their involvement in female reproductive function and a new applicable approach. Vet J 2005;169: 75–84.
66
P.A.R. Hawken et al. / Theriogenology 68 (2007) 56–66
[30] Weems YS, Kim L, Humphreys V, Tsuda V, Weems CW. Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E2 (PGE2) and F2alpha (PGF2alpha) and progesterone in vitro. Prostaglandins Other Lipid Mediat 2003;71:55–73. [31] Yildiz S, Saatci M, Uzun M, Guven B. Effects of ram introduction after the second prostaglandin F2 alpha injection on day 11 on the LH surge characteristics in fat-tailed ewes. Reprod Domest Anim 2003;38:54–7. [32] Yildiz S, Metehan U, Mehmet K. Effects of rams and luteal or follicular phase ewes on preovulatory LH surge characteristics in ewes. Turk J Vet Anim Sci 2004;28:669–73. [33] Ungerfeld R, Carbajal B, Rubianes E, Forsberg M. Endocrine and ovarian changes in response to the ram effect in medrox-
[34]
[36]
[37]
[38]
yprogesterone acetate-primed Corriedale ewes during the breeding and nonbreeding season. Acta Vet Scand 2005;46:33–44. Scaramuzzi RJ, Adams NR, Baird DT, Campbell BK, Downing JA, Findlay JK, et al. A model for follicle selection and the determination of ovulation rate in the ewe. Reprod Fertil Dev 1993;5:459–78. Ngere LO, Dzakuma JM. The effect of sudden introduction of rams on oestrous pattern of tropical ewes. J Agric Sci 1975;84. Chemineau P. Effect on oestrus and ovulation of exposing creole goats to the male at three times of the year. J Reprod Fertil 1983;67:65–72. Skinner DC, Cilliers SD, Skinner JD. Effect of ram introduction on the oestrous cycle of springbok ewes (Antidorcas marsupialis). Reproduction 2002;124:509–13.