- Email: [email protected]
The effect of supplementary long days, a subcutaneous melatonin implant and exposure to a ram on the onset of the breeding season in ewes
Animal Reproduction Science, 34 ( 1994 ) 231-240
231
0378-4320/94/$07.00 © 1994 - Elsevier Science Publishers B.V. All fights reserved
The effect of supplementary long days, a subcutaneous melatonin implant and exposure to a ram on the onset of the breeding season in ewes
A. Donovan, M.P. Boland, J.F. Roche, D. O'CaUaghan* National Agriculturaland VeterinaryBiotechnology Centre, UniversityCollegeDublin, Ireland (Accepted 28 May 1993)
Abstract
Seasonality of breeding in sheep occurs as a consequence of exposure to photoperiodic, social and other environmental stimuli. The objective of this experiment was to determine if the onset of the breeding season in ewes could be advanced by giving supplementary long days during pregnancy, followed by a subcutaneous melatonin implant at lambing. The additional effect of exposing ewes treated in this way to a ram was also investigated. Twin bearing ewes (mean + SEM lambing date 7 March + 2 days) maintained on natural photoperiod were randomly allocated within breed to one of the following treatments: ( 1) control, ewes maintained on natural photoperiod throughout (n = 22 ); (2) melatonin, ewes maintained on natural photoperiod throughout and given a subcutaneous melatonin implant at lambing (n = 23 ); (3) long days plus melatonin, ewes were exposed to 18 h light: 6 h dark for an average of 44 days before lambing, given a melatonin implant at lambing and then maintained on natural photoperiod (n=21); (4) long days plus melatonin plus ram, as Treatment 3, followed by continuous exposure to a ram from the summer solstice (n = 22 ). Reproductive activity was assessed from serum progesterone concentrations in blood collected once weekly from 1 June. Control ewes began their breeding season in September (median date 21 September, range 50 days). Ewes given melatonin alone became reproductively active around the same time (median date 13 September, range 77 days; P> 0.05). Ewes exposed to long days during pregnancy followed by a melatonin implant at lambingbegan their breeding season earlier than controls (median date 6 September, range 93 days; P< 0.05 ). Exposure to a ram from the summer solstice in addition to treatment with long days and melatonin resulted in an additional advancement in the onset of the breeding season (median date 29 July, range 77 days) compared with ewes exposed to long days and given melatonin (P<0.01). Thus, the combined treatment of long days during pregnancy followed by a melatonin implant at lambing can advance the breeding season of ewes. Additional exposure to a ram in such ewes can further advance the onset of the breeding season. *Corresponding author: Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland. Fax. 353-1-6600883.
SSD10378-4320(93)01250-C
232
A. Donovan et al. / Animal Reproduction Science 34 (1994) 231-240
Introduction
Seasonality of breeding in ewes normally results in a single annual pregnancy, limits the lamb output from sheep production systems and reduces the times of the year when high quality lambs are available for sale. Annual changes in photoperiod are key external cues regulating both the timing of the onset and the duration of the breeding season in sheep (Yeates, 1949; Hafez, 1952). While ewes begin breeding in the autumn coincident with shortening daylength, it has been suggested that it is a combination of the time of initial exposure to long (or increasing) daylength, followed by the termination of exposure to long days (normally achieved by exposure to short or decreasing daylength) that is responsible for timing the onset of reproductive activity (Malpaux et al., 1989; Wayne et al., 1990). The specific role of short days in the regulation of this process is to remove the inhibitory effects of long days and also to sustain a breeding season of normal duration (Malpaux and Karsch, 1990; O'Callaghan et al., 1991 a). Thus, for a breeding season to occur in the autumn, the ewe must be exposed to a specific sequence of photoperiodic signals, which consists of long days in spring, followed by a short day photoperiodic signal in late summer/autumn. By mimicking this sequence of photoperiodic signals earlier in the year using controlled photoperiods, it is possible to advance the time of onset of the breeding season in the ewe (O'Callaghan et al., 1992). There are, however, practical constraints associated with photoperiodic manipulation, especially if treatment involves short photoperiod, owing to the problems of housing ewes in summer in light-proof houses, ventilation requirements and cost of indoor feeding. A means of controlling the time of the breeding season may be to combine photoperiodic treatments and exposure to continuous release melatonin implants. Physiological effects of exposure to specific photoperiods are mediated via the secretion of melatonin from the pineal gland. Continuous delivery of melatonin can summate with endogenous melatonin to extend the time each day that melatonin concentrations are elevated in blood (Kennaway et al., 1982a). Exogenous melatonin given in continuous-release preparations maintains elevated concentrations of melatonin in the circulation throughout the 24 h day (O'Callaghan et al., 199 lb; Staples et al., 1992) and is thought to impart an endocrine signal similar to that resulting from exposure to short days (Lincoln and Ebling, 1985; O'Callaghan et al., 199 lb). Much research has focused on administration of exogenous melatonin to mimic short-day photoperiodic cues as a means of advancing the breeding season in anoestrous ewes. Melatonin has been administered in the form of a daily injection (Nett and Niswender, 1982 ), an oral dose (Kennaway et al., 1982b; Arendt et al., 1983), or in a selection of continuous-release forms such as a rumen degradable bolus (Poulton et al., 1987), a vaginal implant (Nowak and Rodway, 1985) or a continuous re-
A. Donovan et aL / Animal Reproduction Science 34 (1994) 231-240
233
lease subcutaneous implant (English et al., 1986; Ronayne et al., 1989). Melatonin given in late anoestrus using these delivery systems can advance the onset of reproductive activity, albeit in many cases the advance in the breeding season is relatively small. Despite the importance of photoperiod in timing reproductive transitions, it has been demonstrated that social stimuli from flockmates can cause changes in the reproductive state of the ewe. The introduction of a ram in late anoestrus to ewes previously isolated from rams can cause ovulation and an earlier onset of breeding activity (Oldham et al., 1978/1979). However, the response to ram introduction depends on the time of the year of ram introduction (Edgar and Bilkey, 1963 ). Such differences in response to social signals from rams may reflect the stage of the annual reproductive cycle of the ewe at the time of treatment. In addition to the acute effects of ram introduction, continuous exposure to rams for up to 1 year can also alter the onset and the end of the breeding season of ewes (Donovan et al., 1991 ). The objectives of this experiment were to determine if the breeding season of ewes could be advanced by providing a sequence of photoperiodic/hormonal signals earlier in the year, to mimic those normally experienced later in the year in natural photoperiod conditions. Specifically, the effect of long days given during late pregnancy (early spring) followed by a subcutaneous melatonin implant at lambing was determined. An additional component of this experiment was to determine if the onset of the breeding season could be further advanced in ewes that were exposed to long days and given a melatonin implant by introducing a ram from the summer solstice. Materials and methods
Animals The experiment was carried out on twin bearing cross-bred ewes between 2 and 5 years of age. Ewes were maintained at pasture (53 °N) until housed on 22 January (44 _ 2 days prior to lambing). Ewes were randomised on a breed basis to one of the following treatments (Fig. 1 ). ( 1 ) Control: ewes maintained on natural photoperiod throughout (n = 22 ). (2) Melatonin: ewes maintained on natural photoperiod throughout and given a subcutaneous implant containing melatonin in a silastic sachet at lambing (n -- 23 ). (3) Long days plus melatonin: ewes were exposed to 18 h light: 6 h dark for 44 days before lambing, given a melatonin implant at lambing and then maintained on natural photoperiod (n = 21 ). (4) Long days plus melatonin plus ram: photoperiod as in Treatment 3, followed by continuous exposure to a ram from the summer solstice (21 June; n=22).
234
A. Donovan et aL / Aniraal Reproduction Science 34 (1994) 231-240
3) Long days plus melatonin (n=21) 44LD
1) Control (n=22) 181_
8.5L
" ~ 8.5L Q.
._~ o ~. -1 18L 0 "1"
. I
MelatoninImplant
I
4) Long days plus melatonin plus ram (n=22) 44LD [ Vasectomized ram ]
2) Melatonin (n=23)
8
[
L t .... Melatonin Implant IDa IFi IAI I jr IAI tOI ID~
8.5L
5
I
I MelaloninImplanl IDr iFi IA= t j i i A, iOi iD1
I
Time of year (months) Fig. 1. Experimental design. The line represents the photoperiod to which the ewes were exposed.
Housed ewes had water and silage available ad libitum and were each fed 0.2 kg day -1 rising in weekly increments of 0.1 kg day-' to 0.7 kg day -x supplementary concentrates during the last 6 weeks of pregnancy. The mean lambing date was 7 March + 2 days. Supplementary long days were regulated by electronic timers which operated fluorescent bulbs producing approximately 350 lx light intensity lateral to the head of ewes. This provision of lighting was in addition to natural photoperiod. Lights were scheduled to come on before dawn (04: 00 h) and remained on until after dark (22: 00 h). Therefore, ewes exposed to supplementary long days received a light signal which was an accumulation of artificial and natural lighting. Melatonin implants contained 700 mg melatonin (Sigma, Poole, UK) and were prepared from silastic sheeting (3.5 era×5.0 cm, 500-1, Dow Coming, Midland, MI) as described by Roche et al. (1985). Implants were inserted subcutaneously in the axillary region, within 12 h of lambing and were recovered from all treated ewes at the end of the experiment, which was after the onset of the next breeding season (more than 200 days after lambing). After lambing, all animals were kept at pasture and isolated by at least 100 m from rams. Ewes to be exposed to a ram (Treatment 4) were maintained in a separate paddock after weaning ( 14 June). One vasectomised ram was kept with this group following introduction during the summer solstice.
Assessment of reproductive activity and data analysis Ovulatory cycles were assessed by measurement of serum progesterone concentrations in blood samples collected once weekly from 1 June. These
235
A. Donovan et aL / Animal Reproduction Science 34 (1994) 231-240
samples were kept at room temperature for 1 h after collection and kept at 4°C overnight. Samples were then centrifuged at 700 × g for 20 min and serum collected and stored at - 2 0 ° C until assays were performed. Progesterone concentrations were determined using the non-extraction radioimmunoassay procedure of Ronayne and Hynes (1990), modified for serum. Validation of the assay for serum involved determining the progesterone concentration in five blood samples collected into untreated or lithium-heparin coated blood collection tubes and serum or plasma was harvested, respectively. The percentage binding of radiolabelled progesterone was not different in serum or plasma in each of the five samples when assayed at five different volumes. Inter-assay coefficients of variation (CV) for three serum pools with mean values of 0.3 ng ml- ~, 1.4 ng ml-1 and 3.2 ng ml-~ averaged 10.9%, 5.5% and 6.2%, respectively. Intra-assay CV for the same three serum pools were 5.0%, 5.2% and 5.1%, respectively. The sensitivity of the assay was 0.2 ng m1-1. The onset of reproductive activity was defined as the date of the first proii
=
[---lq-
o
I
T
I
b
•
- I lstQ
2ndQ"
l
3rdO
" ~th (~
1 O0
~
l---
-
-
m
80
,o ~
20
June
July
August
September I
October
Fig. 2. Upper panel: boxplot showing onset of ovulatory cycles for the four treatment groups: II, control (n = 22); O, melatonin (n = 23 ); 0, long days plus melatonin (n = 21 ); A, long days plus melatonin plus ram (n = 22). The thick vertical line through each box represents the median date of onset of ovulatory cycles, the beginning and end of the box represent the end of the first and third quartile (Q), respectively, and the ends of the horizontal lines represent the minimum and maximum values in each group. Treatments not sharing a common superscript are significantly different (P~< 0.05 ). Lower panel: cumulative percentage of ewes cyclic by date for the four treatment groups (legends as in upper panel).
236
A. Donovan et al. / Animal Reproduction Science 34 (1994) 231-240
gesterone value above 0.5 ng m1-1 in a series of two or more consecutive samples above 0.5 ng ml -~, or one sample above 1 rig m1-1. Differences in the date of onset of reproductive activity were determined using KruskalWallis and Mann-Whitney non-parametric tests. Results The time of onset of reproductive activity (median date, quartile values and range in onset dates) for each group and the percentage of ewes on each treatment that were going through ovulatory cycles plotted against time are presented in Fig. 2. The breeding season in control ewes commenced in September (median date 21 September, range 50 days). The onset of the breeding season was not significantly different in ewes that received melatonin at lambing compared with controls (median date 13 September, range 77 days; P> 0.05 ). Ewes treated with 44 long days during late pregnancy and given a melatonin implant at lambing began their breeding season earlier than control ewes (median date 6 September, range 93 days; P< 0.05 ). The onset of reproductive activity in ewes given long days during late pregnancy, a melatonin implant at lambing and also exposed to a ram from the summer solstice was earlier than in controls (P< 0.0001 ) and earlier than in ewes given long days and a melatonin implant at lambing (median date 29 July, range 77 days; P< 0.01 ). Discussion The main findings from this study were that ewes given long days during late pregnancy followed by a melatonin implant at lambing began their breeding season earlier than controls, and exposure to a ram from the summer solstice resulted in an additional advancement in the onset of the breeding season. Many experiments have demonstrated that administration of melatonin during the summer months can advance the onset of reproductive activity. In contrast, administration of melatonin in spring is ineffective in advancing the onset of the breeding season (English et al., 1986; Williams et al., 1992 ). The results of our study confirm this, as ewes kept on natural photoperiod that were given only a melatonin implant in March did not show a significantly advanced onset of reproductive activity. This suggests that the reproductive response of ewes to melatonin treatment is affected by recent photoperiodic history or the stage of the seasonal reproductive cycle at the time of treatment. This concept is consistent with recent findings that the reproductive response of ewes to a specific photoperiodic manipulation is dependent on the time of year that the photoperiodic challenge is given (O'Callaghan et al., 1992). In contrast to ewes given melatonin alone, ewes that were exposed to long
A. Donovan et al. / Animal Reproduction Science 34 (1994) 231-240
237
days before receiving a melatonin implant commenced their breeding season significantly earlier than controls. Malpaux et al. (1989) has demonstrated a requirement for long days in spring as a prerequisite to synchronise the onset of reproductive activity. The failure of melatonin implantation in March to advance the onset of reproductive activity may result from insufficient exposure to long days prior to melatonin, thus rendering them unresponsive to the subsequent short-day patterns ofmelatonin. Thus, practical manipulation of the annual reproductive cycle of the ewe needs to take into account that a sequence of photoperiodic/hormonal signals will be required at certain times of the annual reproductive cycle, but not at other times of the cycle. When ewes have already received the first part of the signal from natural photoperiod, a single exogenous signal may suffice. This study also indicates that, in addition to supplementary long days followed by a melatonin implant resulting in an advanced breeding season, exposure to a ram from the summer solstice can further advance reproductive activity. The additional response in this group may be related to the fact that ewes treated with long days and melatonin are in a more responsive phase of the annual reproductive cycle; thus the response following ram introduction is enhanced compared with the normal response following ram introduction. Alternatively, this further advancement in the onset of reproductive activity may have resulted directly from the introduction of the ram, regardless of the treatment with long days and melatonin. While it is not possible to differentiate between these two possibilities from this experiment, there was a dramatic effect of a combination of long-day photoperiod, melatonin and exposure to a ram on the onset of the breeding season in ewes. An interesting observation from our study is that the response of ewes to the ram in terms of the rate of onset of ovulatory cycles was not similar to that following acute ram introduction. In the case of ewes responding acutely to ram introduction, peaks of first oestrus normally occur about 18 and 24 days after ram introduction (Martin et al., 1986). In our experiments, the onset of reproductive activity was earlier but at the same rate as in controls and there were no peaks in the number becoming reproductively active over specific days. This suggests that ewes must be close to the natural onset of their breeding season to have synchronous ovulations following ram introduction and that the ram may have been introduced too early in this experiment to observe this effect. The results of our experiment have significant implications for breeding ewes outside the normal breeding season. Our study has demonstrated the requirement for long days as a prerequisite to a positive response from melatonin treatment early in the year. The exact number of long days required appears to be dependent on the phase of the annual reproductive cycle or photoperiodic history, or a combination of these two variables. Recent studies have suggested that between 30 and 60 long days at or before the winter solstice are necessary to induce responsiveness to an inductive photoregimen
238
A. Donovan et al. / Animal Reproduction Science 34 (1994) 231-240
(Jackson et al., 1988; Khalid and Jackson, 1991 ). In addition, Haresign (1992) recommended a 5-6 week interval between melatonin treatment and ram introduction to maximise the effect of novel ram introduction. If the number of long days required to induce onset of reproductive activity in deep anoestrus was known, it should be possible to provide ewes with their complement of long days, a melatonin implant and exposure to rams at an earlier stage in the year. This may induce an even earlier onset of reproductive activity than was obtained in this study. It remains to be determined if ewes induced to become reproductively active earlier in the year from such combined treatments can sustain normal pregnancy rates. In conclusion, melatonin is an effective means of advancing the onset of reproductive activity when given in spring, but only where it is preceded by exposure to long days. Early exposure to rams will further advance the onset of reproductive activity in ewes.
Acknowledgements The authors are grateful to M. Crowe, P. Duffy, F. Griffith, E. Lambert, A. Scanlon and S. Sunderland for technical assistance, G. Claffey, N. Hynes and S. Ni Ceallaigh for laboratory assistance, F. Karsch, The University of Michigan, Ann Arbor for assistance with the design of the experiment, D. Williams for statistical advice, Clonatec Ltd., France for supplying progesterone antibody. This work was supported by the National Agricultural and Veterinary Biotechnology Centre of BioResearch Ireland and University College Dublin, Ireland.
References Arendt, J., Symons, A.M., Laud, C.A. and Pryde, S.J., 1983. Melatonin can induce early onset of the breeding season in ewes. J. Endocrinol., 97: 395-400. Donovan, A., O'Callaghan, D., Boland, M.P., Karsch, F.J. and Roche, J.F., 1991. The relative importance of social signals from ewes and rams in influencing the timing of the breeding season of ewes. J. Reprod. Fertil., Abstr. Ser. 7, Abstr. 30. Edgar, D.G. and Bilkey, D.A., 1963. The influence of rams on the onset of the breeding season in ewes. Proc. N.Z. Soc. Anim. Prod., 23: 79-87. English, J., Poulton, A.L., Arendt, J. and Symons, A.M., 1986. A comparison of the efficiency of melatonin treatments in advancing oestrus in ewes. J. Reprod. Fertil., 77:321-327. Hafez, E.S.E., 1952. Studies on the breeding season and reproduction of the ewe. J. Agric. Sci., 42:189-265. Haresign, W., 1992. Responses of ewes to melatonin implants: importance of the interval between treatment and ram introduction on the synchrony of mating, and effects on ovulation rate. Anim. Prod., 54: 41-45. Jackson, G.L., Gibson, M. and Kuehl, D., 1988. Photoperiodic disruption of photorefractoriness in the ewe. Biol. Reprod., 38: 127-134. Kennaway, D.J., Gilmore, T.A. and Seamark, R.F., 1982a. Effects of melatonin implants on the
A. Donovan et al. / AnimaI Reproduction Science 34 (1994) 231-240
239
circadian rhythm of plasma melatonin and prolactin in sheep. Endocrinology, 110: 21862188. Kennaway, D.J., Gilmore, T.A. and Seamark, R.F., 19821). Effect ofmelatonin feeding on serum prolactin and gonadotropin levels and the onset of seasonal estrous cyclicity in sheep. Endocrinology, 110:1766-1772. Khalid, M. and Jackson, G.L., 1991. Exposure of ewes to long-day photoperiods before the winter solstice can disrupt refractoriness to short days. Anim. Reprod. Sci., 25: 225-232. Lincoln, G.A. and Ebling, F.J.P., 1985. Effect of constant-release implants ofmelatonin on seasonal cycles in reproduction, prolactin secretion and moulting in rams. J. Reprod. Fertil., 73: 241-253. Malpaux, B. and Karsch, F.J., 1990. A role for short clays in sustaining seasonal reproductive activity in the ewe. J. Reprod. Fertil., 90: 555-562. Malpaux, B., Robinson, J.E., Wayne, N.L. and Karsch, F.J., 1989. Regulation of the onset of the breeding season of the ewe: Importance of long clays and of an endogenous reproductive rhythm. J. Endocrinol., 122: 269-278. Martin, G.B., Oldham, C.M., Cognie, Y. and Pearce, D.T., 1986. The physiological responses of anovulatory ewes to the introduction of rams---a review. Livest. Prod. Sci., 15:219-247. Nett, T.M. and Niswender, G.D., 1982. Influence of exogenous melatonin on seasonality of reproduction in sheep. Theriogenology, 17: 645-653. Nowak, R. and Rodway, R.G., 1985. Effects of intravaginal implants of melatonin on the onset of ovarian activity in adult and prepubertal ewes. J. Reprod. Fertil., 74: 287-293. O'Callaghan, D., Karsch, F.J., Boland, M.P. and Roche, J.F., 1991 a. Role of short days in timing the onset and duration of reproductive activity in ewes under artificial photoperiods. Biol. Reprod., 44: 23-28. O'Callaghan, D., Karsch, F.J., Boland, M.P. and Roche, J.F., 199 lb. What photoperiodic signal is provided by a continuous-release melatonin implant? Biol. Reprod., 45: 927-933. O'Callaghan, D., Donovan, A., Karsch, F.J., Boland, M.P. and Roche, J.F., 1992. Reproductive response of ewes to a specific photoperiodic challenge at different times of the year. In: Proc. Third Meeting of Society for Research on Biological Rhythms, Amelia Island, Jacksonville, FL, 6-10 May 1992, Abstr. 68. Oldham, C.M., Martin, G.B. and Knight, T.W., 1978/1979. Stimulation of seasonally anovular Merino ewes by rams. I. Time from introduction of the rams to the preovulatory LH surge and ovulation. Anim. Reprod. Sci., 1: 283-290. Poulton, A.L., Symons, A.M., Kelly, M.I. and Arendt, J., 1987. Intraruminal soluble glass boluses containing melatonin can induce early onset of ovarian activity in ewes. J. Reprod. Ferfil., 80: 235-239. Roche, J.F., Hanrahan, J.P., Quirke, J.F. and Ronayne, E., 1985. Effects of melatonin on the time of onset of the breeding season in different breeds of sheep. In: F. Ellendorff and F. Elsaesser (Editor), Endocrine Causes of Seasonal and Lactational Anoestrus in Farm Animals. Martinus Nijhoff, Dordrecht, pp. 55-65. Ronayne, E. and Hynes, N., 1990. Measurement of plasma progesterone concentrations by extraction and non-extraction radioimmunoassays. It. J. Agric. Res., 29:109-115. Ronayne, E., Jordan, B., Quirke, J.F. and Roche, J.F., 1989. The effect of frequency of administration of melatonin on the time of onset of the breeding season in anoestrous ewes. Anim. Reprod. Sci., 18: 13-24. Staples, L.D., McPhee, S., Kennaway, D.J. and Williams, A.H., 1992. The influence of exogenous melatonin on the seasonal patterns of ovulation and oestrus in sheep. Anim. Reprod. Sci., 30: 185-223. Wayne, N.L., Malpaux, B. and Karsch, F.J., 1990. Photoperiodic requirements for timing onset and duration of the breeding season in the ewe. J. Comp. Physiol. A, 166: 835-842. Williams, A.H., McPhee, S.R., Reeve, J.L. and Staples, L.D., 1992. Optimum use of subcuta-
240
A. Donovan et al. / Animal Reproduction Science 34 (1994) 231-240
neous melatonin implants to enhance the reproductive performance of seasonal and nonseasonal sheep joined in spring and early summer. Anim. Reprod. Sci., 30: 225-258. Yeates, N.T.M., 1949. The breeding season of the sheep with particular reference to its modification by artificial means using light. J. Agric. Sci., 39: 1-43.
231
0378-4320/94/$07.00 © 1994 - Elsevier Science Publishers B.V. All fights reserved
The effect of supplementary long days, a subcutaneous melatonin implant and exposure to a ram on the onset of the breeding season in ewes
A. Donovan, M.P. Boland, J.F. Roche, D. O'CaUaghan* National Agriculturaland VeterinaryBiotechnology Centre, UniversityCollegeDublin, Ireland (Accepted 28 May 1993)
Abstract
Seasonality of breeding in sheep occurs as a consequence of exposure to photoperiodic, social and other environmental stimuli. The objective of this experiment was to determine if the onset of the breeding season in ewes could be advanced by giving supplementary long days during pregnancy, followed by a subcutaneous melatonin implant at lambing. The additional effect of exposing ewes treated in this way to a ram was also investigated. Twin bearing ewes (mean + SEM lambing date 7 March + 2 days) maintained on natural photoperiod were randomly allocated within breed to one of the following treatments: ( 1) control, ewes maintained on natural photoperiod throughout (n = 22 ); (2) melatonin, ewes maintained on natural photoperiod throughout and given a subcutaneous melatonin implant at lambing (n = 23 ); (3) long days plus melatonin, ewes were exposed to 18 h light: 6 h dark for an average of 44 days before lambing, given a melatonin implant at lambing and then maintained on natural photoperiod (n=21); (4) long days plus melatonin plus ram, as Treatment 3, followed by continuous exposure to a ram from the summer solstice (n = 22 ). Reproductive activity was assessed from serum progesterone concentrations in blood collected once weekly from 1 June. Control ewes began their breeding season in September (median date 21 September, range 50 days). Ewes given melatonin alone became reproductively active around the same time (median date 13 September, range 77 days; P> 0.05). Ewes exposed to long days during pregnancy followed by a melatonin implant at lambingbegan their breeding season earlier than controls (median date 6 September, range 93 days; P< 0.05 ). Exposure to a ram from the summer solstice in addition to treatment with long days and melatonin resulted in an additional advancement in the onset of the breeding season (median date 29 July, range 77 days) compared with ewes exposed to long days and given melatonin (P<0.01). Thus, the combined treatment of long days during pregnancy followed by a melatonin implant at lambing can advance the breeding season of ewes. Additional exposure to a ram in such ewes can further advance the onset of the breeding season. *Corresponding author: Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland. Fax. 353-1-6600883.
SSD10378-4320(93)01250-C
232
A. Donovan et al. / Animal Reproduction Science 34 (1994) 231-240
Introduction
Seasonality of breeding in ewes normally results in a single annual pregnancy, limits the lamb output from sheep production systems and reduces the times of the year when high quality lambs are available for sale. Annual changes in photoperiod are key external cues regulating both the timing of the onset and the duration of the breeding season in sheep (Yeates, 1949; Hafez, 1952). While ewes begin breeding in the autumn coincident with shortening daylength, it has been suggested that it is a combination of the time of initial exposure to long (or increasing) daylength, followed by the termination of exposure to long days (normally achieved by exposure to short or decreasing daylength) that is responsible for timing the onset of reproductive activity (Malpaux et al., 1989; Wayne et al., 1990). The specific role of short days in the regulation of this process is to remove the inhibitory effects of long days and also to sustain a breeding season of normal duration (Malpaux and Karsch, 1990; O'Callaghan et al., 1991 a). Thus, for a breeding season to occur in the autumn, the ewe must be exposed to a specific sequence of photoperiodic signals, which consists of long days in spring, followed by a short day photoperiodic signal in late summer/autumn. By mimicking this sequence of photoperiodic signals earlier in the year using controlled photoperiods, it is possible to advance the time of onset of the breeding season in the ewe (O'Callaghan et al., 1992). There are, however, practical constraints associated with photoperiodic manipulation, especially if treatment involves short photoperiod, owing to the problems of housing ewes in summer in light-proof houses, ventilation requirements and cost of indoor feeding. A means of controlling the time of the breeding season may be to combine photoperiodic treatments and exposure to continuous release melatonin implants. Physiological effects of exposure to specific photoperiods are mediated via the secretion of melatonin from the pineal gland. Continuous delivery of melatonin can summate with endogenous melatonin to extend the time each day that melatonin concentrations are elevated in blood (Kennaway et al., 1982a). Exogenous melatonin given in continuous-release preparations maintains elevated concentrations of melatonin in the circulation throughout the 24 h day (O'Callaghan et al., 199 lb; Staples et al., 1992) and is thought to impart an endocrine signal similar to that resulting from exposure to short days (Lincoln and Ebling, 1985; O'Callaghan et al., 199 lb). Much research has focused on administration of exogenous melatonin to mimic short-day photoperiodic cues as a means of advancing the breeding season in anoestrous ewes. Melatonin has been administered in the form of a daily injection (Nett and Niswender, 1982 ), an oral dose (Kennaway et al., 1982b; Arendt et al., 1983), or in a selection of continuous-release forms such as a rumen degradable bolus (Poulton et al., 1987), a vaginal implant (Nowak and Rodway, 1985) or a continuous re-
A. Donovan et aL / Animal Reproduction Science 34 (1994) 231-240
233
lease subcutaneous implant (English et al., 1986; Ronayne et al., 1989). Melatonin given in late anoestrus using these delivery systems can advance the onset of reproductive activity, albeit in many cases the advance in the breeding season is relatively small. Despite the importance of photoperiod in timing reproductive transitions, it has been demonstrated that social stimuli from flockmates can cause changes in the reproductive state of the ewe. The introduction of a ram in late anoestrus to ewes previously isolated from rams can cause ovulation and an earlier onset of breeding activity (Oldham et al., 1978/1979). However, the response to ram introduction depends on the time of the year of ram introduction (Edgar and Bilkey, 1963 ). Such differences in response to social signals from rams may reflect the stage of the annual reproductive cycle of the ewe at the time of treatment. In addition to the acute effects of ram introduction, continuous exposure to rams for up to 1 year can also alter the onset and the end of the breeding season of ewes (Donovan et al., 1991 ). The objectives of this experiment were to determine if the breeding season of ewes could be advanced by providing a sequence of photoperiodic/hormonal signals earlier in the year, to mimic those normally experienced later in the year in natural photoperiod conditions. Specifically, the effect of long days given during late pregnancy (early spring) followed by a subcutaneous melatonin implant at lambing was determined. An additional component of this experiment was to determine if the onset of the breeding season could be further advanced in ewes that were exposed to long days and given a melatonin implant by introducing a ram from the summer solstice. Materials and methods
Animals The experiment was carried out on twin bearing cross-bred ewes between 2 and 5 years of age. Ewes were maintained at pasture (53 °N) until housed on 22 January (44 _ 2 days prior to lambing). Ewes were randomised on a breed basis to one of the following treatments (Fig. 1 ). ( 1 ) Control: ewes maintained on natural photoperiod throughout (n = 22 ). (2) Melatonin: ewes maintained on natural photoperiod throughout and given a subcutaneous implant containing melatonin in a silastic sachet at lambing (n -- 23 ). (3) Long days plus melatonin: ewes were exposed to 18 h light: 6 h dark for 44 days before lambing, given a melatonin implant at lambing and then maintained on natural photoperiod (n = 21 ). (4) Long days plus melatonin plus ram: photoperiod as in Treatment 3, followed by continuous exposure to a ram from the summer solstice (21 June; n=22).
234
A. Donovan et aL / Aniraal Reproduction Science 34 (1994) 231-240
3) Long days plus melatonin (n=21) 44LD
1) Control (n=22) 181_
8.5L
" ~ 8.5L Q.
._~ o ~. -1 18L 0 "1"
. I
MelatoninImplant
I
4) Long days plus melatonin plus ram (n=22) 44LD [ Vasectomized ram ]
2) Melatonin (n=23)
8
[
L t .... Melatonin Implant IDa IFi IAI I jr IAI tOI ID~
8.5L
5
I
I MelaloninImplanl IDr iFi IA= t j i i A, iOi iD1
I
Time of year (months) Fig. 1. Experimental design. The line represents the photoperiod to which the ewes were exposed.
Housed ewes had water and silage available ad libitum and were each fed 0.2 kg day -1 rising in weekly increments of 0.1 kg day-' to 0.7 kg day -x supplementary concentrates during the last 6 weeks of pregnancy. The mean lambing date was 7 March + 2 days. Supplementary long days were regulated by electronic timers which operated fluorescent bulbs producing approximately 350 lx light intensity lateral to the head of ewes. This provision of lighting was in addition to natural photoperiod. Lights were scheduled to come on before dawn (04: 00 h) and remained on until after dark (22: 00 h). Therefore, ewes exposed to supplementary long days received a light signal which was an accumulation of artificial and natural lighting. Melatonin implants contained 700 mg melatonin (Sigma, Poole, UK) and were prepared from silastic sheeting (3.5 era×5.0 cm, 500-1, Dow Coming, Midland, MI) as described by Roche et al. (1985). Implants were inserted subcutaneously in the axillary region, within 12 h of lambing and were recovered from all treated ewes at the end of the experiment, which was after the onset of the next breeding season (more than 200 days after lambing). After lambing, all animals were kept at pasture and isolated by at least 100 m from rams. Ewes to be exposed to a ram (Treatment 4) were maintained in a separate paddock after weaning ( 14 June). One vasectomised ram was kept with this group following introduction during the summer solstice.
Assessment of reproductive activity and data analysis Ovulatory cycles were assessed by measurement of serum progesterone concentrations in blood samples collected once weekly from 1 June. These
235
A. Donovan et aL / Animal Reproduction Science 34 (1994) 231-240
samples were kept at room temperature for 1 h after collection and kept at 4°C overnight. Samples were then centrifuged at 700 × g for 20 min and serum collected and stored at - 2 0 ° C until assays were performed. Progesterone concentrations were determined using the non-extraction radioimmunoassay procedure of Ronayne and Hynes (1990), modified for serum. Validation of the assay for serum involved determining the progesterone concentration in five blood samples collected into untreated or lithium-heparin coated blood collection tubes and serum or plasma was harvested, respectively. The percentage binding of radiolabelled progesterone was not different in serum or plasma in each of the five samples when assayed at five different volumes. Inter-assay coefficients of variation (CV) for three serum pools with mean values of 0.3 ng ml- ~, 1.4 ng ml-1 and 3.2 ng ml-~ averaged 10.9%, 5.5% and 6.2%, respectively. Intra-assay CV for the same three serum pools were 5.0%, 5.2% and 5.1%, respectively. The sensitivity of the assay was 0.2 ng m1-1. The onset of reproductive activity was defined as the date of the first proii
=
[---lq-
o
I
T
I
b
•
- I lstQ
2ndQ"
l
3rdO
" ~th (~
1 O0
~
l---
-
-
m
80
,o ~
20
June
July
August
September I
October
Fig. 2. Upper panel: boxplot showing onset of ovulatory cycles for the four treatment groups: II, control (n = 22); O, melatonin (n = 23 ); 0, long days plus melatonin (n = 21 ); A, long days plus melatonin plus ram (n = 22). The thick vertical line through each box represents the median date of onset of ovulatory cycles, the beginning and end of the box represent the end of the first and third quartile (Q), respectively, and the ends of the horizontal lines represent the minimum and maximum values in each group. Treatments not sharing a common superscript are significantly different (P~< 0.05 ). Lower panel: cumulative percentage of ewes cyclic by date for the four treatment groups (legends as in upper panel).
236
A. Donovan et al. / Animal Reproduction Science 34 (1994) 231-240
gesterone value above 0.5 ng m1-1 in a series of two or more consecutive samples above 0.5 ng ml -~, or one sample above 1 rig m1-1. Differences in the date of onset of reproductive activity were determined using KruskalWallis and Mann-Whitney non-parametric tests. Results The time of onset of reproductive activity (median date, quartile values and range in onset dates) for each group and the percentage of ewes on each treatment that were going through ovulatory cycles plotted against time are presented in Fig. 2. The breeding season in control ewes commenced in September (median date 21 September, range 50 days). The onset of the breeding season was not significantly different in ewes that received melatonin at lambing compared with controls (median date 13 September, range 77 days; P> 0.05 ). Ewes treated with 44 long days during late pregnancy and given a melatonin implant at lambing began their breeding season earlier than control ewes (median date 6 September, range 93 days; P< 0.05 ). The onset of reproductive activity in ewes given long days during late pregnancy, a melatonin implant at lambing and also exposed to a ram from the summer solstice was earlier than in controls (P< 0.0001 ) and earlier than in ewes given long days and a melatonin implant at lambing (median date 29 July, range 77 days; P< 0.01 ). Discussion The main findings from this study were that ewes given long days during late pregnancy followed by a melatonin implant at lambing began their breeding season earlier than controls, and exposure to a ram from the summer solstice resulted in an additional advancement in the onset of the breeding season. Many experiments have demonstrated that administration of melatonin during the summer months can advance the onset of reproductive activity. In contrast, administration of melatonin in spring is ineffective in advancing the onset of the breeding season (English et al., 1986; Williams et al., 1992 ). The results of our study confirm this, as ewes kept on natural photoperiod that were given only a melatonin implant in March did not show a significantly advanced onset of reproductive activity. This suggests that the reproductive response of ewes to melatonin treatment is affected by recent photoperiodic history or the stage of the seasonal reproductive cycle at the time of treatment. This concept is consistent with recent findings that the reproductive response of ewes to a specific photoperiodic manipulation is dependent on the time of year that the photoperiodic challenge is given (O'Callaghan et al., 1992). In contrast to ewes given melatonin alone, ewes that were exposed to long
A. Donovan et al. / Animal Reproduction Science 34 (1994) 231-240
237
days before receiving a melatonin implant commenced their breeding season significantly earlier than controls. Malpaux et al. (1989) has demonstrated a requirement for long days in spring as a prerequisite to synchronise the onset of reproductive activity. The failure of melatonin implantation in March to advance the onset of reproductive activity may result from insufficient exposure to long days prior to melatonin, thus rendering them unresponsive to the subsequent short-day patterns ofmelatonin. Thus, practical manipulation of the annual reproductive cycle of the ewe needs to take into account that a sequence of photoperiodic/hormonal signals will be required at certain times of the annual reproductive cycle, but not at other times of the cycle. When ewes have already received the first part of the signal from natural photoperiod, a single exogenous signal may suffice. This study also indicates that, in addition to supplementary long days followed by a melatonin implant resulting in an advanced breeding season, exposure to a ram from the summer solstice can further advance reproductive activity. The additional response in this group may be related to the fact that ewes treated with long days and melatonin are in a more responsive phase of the annual reproductive cycle; thus the response following ram introduction is enhanced compared with the normal response following ram introduction. Alternatively, this further advancement in the onset of reproductive activity may have resulted directly from the introduction of the ram, regardless of the treatment with long days and melatonin. While it is not possible to differentiate between these two possibilities from this experiment, there was a dramatic effect of a combination of long-day photoperiod, melatonin and exposure to a ram on the onset of the breeding season in ewes. An interesting observation from our study is that the response of ewes to the ram in terms of the rate of onset of ovulatory cycles was not similar to that following acute ram introduction. In the case of ewes responding acutely to ram introduction, peaks of first oestrus normally occur about 18 and 24 days after ram introduction (Martin et al., 1986). In our experiments, the onset of reproductive activity was earlier but at the same rate as in controls and there were no peaks in the number becoming reproductively active over specific days. This suggests that ewes must be close to the natural onset of their breeding season to have synchronous ovulations following ram introduction and that the ram may have been introduced too early in this experiment to observe this effect. The results of our experiment have significant implications for breeding ewes outside the normal breeding season. Our study has demonstrated the requirement for long days as a prerequisite to a positive response from melatonin treatment early in the year. The exact number of long days required appears to be dependent on the phase of the annual reproductive cycle or photoperiodic history, or a combination of these two variables. Recent studies have suggested that between 30 and 60 long days at or before the winter solstice are necessary to induce responsiveness to an inductive photoregimen
238
A. Donovan et al. / Animal Reproduction Science 34 (1994) 231-240
(Jackson et al., 1988; Khalid and Jackson, 1991 ). In addition, Haresign (1992) recommended a 5-6 week interval between melatonin treatment and ram introduction to maximise the effect of novel ram introduction. If the number of long days required to induce onset of reproductive activity in deep anoestrus was known, it should be possible to provide ewes with their complement of long days, a melatonin implant and exposure to rams at an earlier stage in the year. This may induce an even earlier onset of reproductive activity than was obtained in this study. It remains to be determined if ewes induced to become reproductively active earlier in the year from such combined treatments can sustain normal pregnancy rates. In conclusion, melatonin is an effective means of advancing the onset of reproductive activity when given in spring, but only where it is preceded by exposure to long days. Early exposure to rams will further advance the onset of reproductive activity in ewes.
Acknowledgements The authors are grateful to M. Crowe, P. Duffy, F. Griffith, E. Lambert, A. Scanlon and S. Sunderland for technical assistance, G. Claffey, N. Hynes and S. Ni Ceallaigh for laboratory assistance, F. Karsch, The University of Michigan, Ann Arbor for assistance with the design of the experiment, D. Williams for statistical advice, Clonatec Ltd., France for supplying progesterone antibody. This work was supported by the National Agricultural and Veterinary Biotechnology Centre of BioResearch Ireland and University College Dublin, Ireland.
References Arendt, J., Symons, A.M., Laud, C.A. and Pryde, S.J., 1983. Melatonin can induce early onset of the breeding season in ewes. J. Endocrinol., 97: 395-400. Donovan, A., O'Callaghan, D., Boland, M.P., Karsch, F.J. and Roche, J.F., 1991. The relative importance of social signals from ewes and rams in influencing the timing of the breeding season of ewes. J. Reprod. Fertil., Abstr. Ser. 7, Abstr. 30. Edgar, D.G. and Bilkey, D.A., 1963. The influence of rams on the onset of the breeding season in ewes. Proc. N.Z. Soc. Anim. Prod., 23: 79-87. English, J., Poulton, A.L., Arendt, J. and Symons, A.M., 1986. A comparison of the efficiency of melatonin treatments in advancing oestrus in ewes. J. Reprod. Fertil., 77:321-327. Hafez, E.S.E., 1952. Studies on the breeding season and reproduction of the ewe. J. Agric. Sci., 42:189-265. Haresign, W., 1992. Responses of ewes to melatonin implants: importance of the interval between treatment and ram introduction on the synchrony of mating, and effects on ovulation rate. Anim. Prod., 54: 41-45. Jackson, G.L., Gibson, M. and Kuehl, D., 1988. Photoperiodic disruption of photorefractoriness in the ewe. Biol. Reprod., 38: 127-134. Kennaway, D.J., Gilmore, T.A. and Seamark, R.F., 1982a. Effects of melatonin implants on the
A. Donovan et al. / AnimaI Reproduction Science 34 (1994) 231-240
239
circadian rhythm of plasma melatonin and prolactin in sheep. Endocrinology, 110: 21862188. Kennaway, D.J., Gilmore, T.A. and Seamark, R.F., 19821). Effect ofmelatonin feeding on serum prolactin and gonadotropin levels and the onset of seasonal estrous cyclicity in sheep. Endocrinology, 110:1766-1772. Khalid, M. and Jackson, G.L., 1991. Exposure of ewes to long-day photoperiods before the winter solstice can disrupt refractoriness to short days. Anim. Reprod. Sci., 25: 225-232. Lincoln, G.A. and Ebling, F.J.P., 1985. Effect of constant-release implants ofmelatonin on seasonal cycles in reproduction, prolactin secretion and moulting in rams. J. Reprod. Fertil., 73: 241-253. Malpaux, B. and Karsch, F.J., 1990. A role for short clays in sustaining seasonal reproductive activity in the ewe. J. Reprod. Fertil., 90: 555-562. Malpaux, B., Robinson, J.E., Wayne, N.L. and Karsch, F.J., 1989. Regulation of the onset of the breeding season of the ewe: Importance of long clays and of an endogenous reproductive rhythm. J. Endocrinol., 122: 269-278. Martin, G.B., Oldham, C.M., Cognie, Y. and Pearce, D.T., 1986. The physiological responses of anovulatory ewes to the introduction of rams---a review. Livest. Prod. Sci., 15:219-247. Nett, T.M. and Niswender, G.D., 1982. Influence of exogenous melatonin on seasonality of reproduction in sheep. Theriogenology, 17: 645-653. Nowak, R. and Rodway, R.G., 1985. Effects of intravaginal implants of melatonin on the onset of ovarian activity in adult and prepubertal ewes. J. Reprod. Fertil., 74: 287-293. O'Callaghan, D., Karsch, F.J., Boland, M.P. and Roche, J.F., 1991 a. Role of short days in timing the onset and duration of reproductive activity in ewes under artificial photoperiods. Biol. Reprod., 44: 23-28. O'Callaghan, D., Karsch, F.J., Boland, M.P. and Roche, J.F., 199 lb. What photoperiodic signal is provided by a continuous-release melatonin implant? Biol. Reprod., 45: 927-933. O'Callaghan, D., Donovan, A., Karsch, F.J., Boland, M.P. and Roche, J.F., 1992. Reproductive response of ewes to a specific photoperiodic challenge at different times of the year. In: Proc. Third Meeting of Society for Research on Biological Rhythms, Amelia Island, Jacksonville, FL, 6-10 May 1992, Abstr. 68. Oldham, C.M., Martin, G.B. and Knight, T.W., 1978/1979. Stimulation of seasonally anovular Merino ewes by rams. I. Time from introduction of the rams to the preovulatory LH surge and ovulation. Anim. Reprod. Sci., 1: 283-290. Poulton, A.L., Symons, A.M., Kelly, M.I. and Arendt, J., 1987. Intraruminal soluble glass boluses containing melatonin can induce early onset of ovarian activity in ewes. J. Reprod. Ferfil., 80: 235-239. Roche, J.F., Hanrahan, J.P., Quirke, J.F. and Ronayne, E., 1985. Effects of melatonin on the time of onset of the breeding season in different breeds of sheep. In: F. Ellendorff and F. Elsaesser (Editor), Endocrine Causes of Seasonal and Lactational Anoestrus in Farm Animals. Martinus Nijhoff, Dordrecht, pp. 55-65. Ronayne, E. and Hynes, N., 1990. Measurement of plasma progesterone concentrations by extraction and non-extraction radioimmunoassays. It. J. Agric. Res., 29:109-115. Ronayne, E., Jordan, B., Quirke, J.F. and Roche, J.F., 1989. The effect of frequency of administration of melatonin on the time of onset of the breeding season in anoestrous ewes. Anim. Reprod. Sci., 18: 13-24. Staples, L.D., McPhee, S., Kennaway, D.J. and Williams, A.H., 1992. The influence of exogenous melatonin on the seasonal patterns of ovulation and oestrus in sheep. Anim. Reprod. Sci., 30: 185-223. Wayne, N.L., Malpaux, B. and Karsch, F.J., 1990. Photoperiodic requirements for timing onset and duration of the breeding season in the ewe. J. Comp. Physiol. A, 166: 835-842. Williams, A.H., McPhee, S.R., Reeve, J.L. and Staples, L.D., 1992. Optimum use of subcuta-
240
A. Donovan et al. / Animal Reproduction Science 34 (1994) 231-240
neous melatonin implants to enhance the reproductive performance of seasonal and nonseasonal sheep joined in spring and early summer. Anim. Reprod. Sci., 30: 225-258. Yeates, N.T.M., 1949. The breeding season of the sheep with particular reference to its modification by artificial means using light. J. Agric. Sci., 39: 1-43.