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Regular Article |
a Department of Clinical Veterinary Science, University of Bristol, Langford House, Langford BS40 5DU, United Kingdom
b Veterinary Wildlife Unit, Faculty of Veterinary Science, Onderstepoort 0110, South Africa
c Institut National de la Recherche Agronomique, Physiologie de la Reproduction des Mammiferes Domestiques, 37380 Nouzilly, France
ABSTRACT
The springbok is an arid-adapted antelope inhabiting the desert and semidesert regions of southern Africa. Because it thrives in these sparsely vegetated areas, the springbok is of potential agricultural importance and the prospect of domestication has been speculated for many years. However, apart from observational studies on its breeding in the wild, suggesting it is an aseasonal breeder, little is known about the underlying reproductive endocrinology of this species. In this study, biweekly peripheral blood samples were collected from eight captive springbok ewes from October 1995 until September 1998 and analyzed for progesterone. At the start of the study, six ewes were prepubertal and cycling commenced spontaneously between November 1995 and June 1996. Cycling had already commenced in two ewes. At the end of November 1996, estrous cycles ceased abruptly in all ewes and restarted in April 1997. Cycling ceased again between December 1997 and February 1998 and restarted in June 1998 in six ewes; there was no cessation of estrous cycles in two ewes. Thus, although some individuals cycle continuously, there is a clear endocrine anestrus of between 4 and 5 mo in springbok, the timing and duration of which is synchronized between some individuals but the time of onset and cessation is variable from year to year. To ensure that the fluctuations we observed in progesterone levels were reliable indicators of changes in the estrous cycle, blood samples were collected every 6 h for 16 days in August 1998. A surge in LH secretion was observed in all ewes 55 ± 5 h after the fall in progesterone. Progesterone levels increased again 45 ± 8 h after the surge. A final study showed that the pattern of melatonin release in springbok exhibits a normal day/night profile, and thus photoperiodic information is transformed into an endocrine code to springbok but does not appear to affect reproduction. Rather, our data raise the possibility that the prevailing ambient temperature may influence the onset of ovarian activity in this species.
ovulatory cycle, pineal, seasonal reproduction
INTRODUCTION
The springbok, Antidorcas marsupialis, is an arid-adapted gazelle confined to southwestern Africa [1]. In common with most wild antelope, little is known about reproduction in this species, not least because of the harsh environment it inhabits and its tendency to disperse, making prolonged observations in the wild impractical. Pressures exerted on animals inhabiting arid environments are extreme and any increment can lead to extinction in the wild, as occurred with the mhorr gazelle, Gazella dama mhorr [2], the Arabian oryx, Oryx leucoryx [3], and the scimitar-horned oryx, Oryx dammah [4]. Indeed, ex situ breeding programs have been initiated on several arid-environment inhabiting species, although basic data remain limited and, to our knowledge, nothing has been published on reproductive cycles in gazelles. Not surprisingly, the optimal periods for manipulating fertility remain largely unidentified and artificial insemination programs have met with limited success [4]; in springbok, a single reported artificial insemination attempt was unsuccessful [5] and, in the mhorr gazelle, 4 of 13 artificially inseminated ewes became pregnant, although only 1 carried the fetus to term [6]. Understanding the reproductive endocrine profile will improve our ability to conserve a species in the wild and, with an increased commercial market for venison, particularly from springbok, knowledge of reproduction will also assist in their husbandry.
To our knowledge, only a single other long-term study over more than a year on individual wild antelope has been reported [7] and most studies concluding that a species breeds aseasonally have relied on the distribution of births. In the wild, it is probable that a ewe will conceive at the first cycle, and thus it is also not possible to establish whether individuals or populations have periods of reproductive quiescence or, in the event of nonconception, whether estrous cycles continue unabated throughout the year. For this, it is essential to carry out studies in the absence of males and, to allow for repetitive sampling of specific individuals, in captivity. Thus, the first study investigated the duration and occurrence of estrous cycles in a captive population of springbok over 30 mo. We hypothesized that as in the more equatorially located dik-dik, Madoqua guentheri [7], and sable antelope, Hippotragus niger [8], these springbok would either remain reproductively active throughout the year or would exhibit unsynchronized periods of reproductive activity with reproductive quiescence, as found in some sheep, Ovis aries, on a fixed photoperiod [9]. Either of these hypotheses would explain the field observations of lambs being born throughout the year (i.e., aseasonality).
There is considerable evidence that photoperiodic information is the primary proximate factor determining the periods of sexual activity and quiescence in seasonal breeders (see reviews [10, 11]). In the sheep, photoperiodic information is transduced to the reproductive neuroendocrine axis by the nocturnal increase in the pineal hormone, melatonin. In an aseasonal species it is possible that melatonin secretion is impaired and that daylight, therefore, does not get transformed into a biochemical signal. To address this question, a further study investigated whether springbok secreted melatonin in accordance with a normal day (low melatonin):night (high melatonin) profile.
MATERIALS AND METHODS
Animals and Husbandry
Juvenile springbok ewes (2–3 mo; n = 8) were captured at the same time near Hutchinson, South Africa, and held in a paddock and hand-reared on cow's milk for 3 mo before being weaned onto alfalfa. At approximately 6 mo of age, the springbok were transferred to the experimental farm of the University of Pretoria, Pretoria (25°S, 28°E) in a 500 m2 enclosure with 3-m-high sides. They were fed daily on alfalfa, cottonseed, and antelope pellets (Epol Ltd, Pretoria, South Africa). This diet was supplemented with freshly cut Salix mucronata, Morusmesozygia leaf, or both, and mineral licks (Kynoch, South Africa). Water was available ad libitum.
All the springbok were released at the end of the study onto a game farm and have since bred successfully.
Blood Sampling
For collection of blood samples, a collapsible chute within the enclosure was opened and the springbok were confined into a small space where they were manually restrained. Blood was taken by venipuncture from the jugular vein and collected into heparinized venoject tubes (Becton Dickinson, Meylan Cedex, France) and kept on ice for 30 min until centrifuged. Plasma was harvested and frozen at -20°C for later radioimmunoassay analysis. In the first study (November 1995–July 1998), sampling was undertaken biweekly between 0800 and 1000 h. To ensure that a preovulatory LH surge was occurring, blood samples were collected every 6 h for 16 days and to investigate whether springbok expressed a nocturnal melatonin increment, blood samples were collected every 4 h and every 2 h at dusk and dawn.
It took approximately 20–25 min to collect samples from all eight springbok.
Radioimmunoassays
Tests for parallelism between springbok plasma and the respective assay standards were carried out on all assays prior to analysis. Specifically, serial dilutions of samples with elevated hormone concentrations yielded curves parallel to those obtained with hormone standards.
The progesterone level in biweekly plasma samples was established using an immunologic radioimmunoassay [12], which revealed whether the plasma sample progesterone concentration was above 1 ng/ml. This assay, which has been used extensively in the sheep, goat, and cow is semiquantitative and several quality controls, particularly around 1 ng/ml, were used. We verified that the distribution of counts per minute (cpm) for all springbok plasma samples was bimodal (populations of samples with low value = high progesterone, and high value = low progesterone) and that the cpm value corresponding to the 1 ng/ml quality control allowed us to discriminate between these two populations. To eliminate intraindividual variation with time, all samples from a ewe were analyzed in the same assay.
For the more frequent blood sampling, progesterone levels were estimated in a single immunometric radioimmunoassay [13]. The intraassay coefficient of variation was 10% and assay sensitivity was 0.05 ng/ml. The standard in both progesterone assays was pure progesterone (Steraloids Inc., Newport, RI).
The 6-h blood samples were assayed for LH in duplicate 100-µl aliquots of plasma using the radioimmunoassay method of Pelletier et al. [14] as modified by Montgomery et al. [15], and all samples were tested in a single assay. The intraassay coefficient of variation averaged 6% and sensitivity was 0.15 ng/ml of standard 1051-CY-LH (1 ng = 2.08 ng NIH-LH-S1; a gift of Y. Combarnous, INRA, France).
Jugular plasma melatonin concentrations were determined in 100-µl aliquots using a single-well-validated radioimmunoassay [16]. Assay sensitivity averaged 16 pg/ml and the intra-assay coefficient of variation was 4%. Standards were made from pure melatonin (Fluka, Saint-Quentin Fallavier, France).
Analysis
The immunologic assay was used to determine whether a ewe was in the follicular or luteal phase of the estrous cycle and also whether the ewe was in the breeding season or anestrus. Specifically, if progesterone concentrations were below 1 ng/ml a ewe was noted to be in the follicular phase, but if concentrations were above 1 ng/ml for at least two sequential samples the ewe was recorded to be in the luteal phase of the estrous cycle. The first sample to exceed 1 ng/ml was recorded as the start of the luteal phase and that ovulation had occurred. If progesterone levels remained basal for more than a month, the ewe was noted as anestrus. To estimate the mean duration of ovarian cycles for the group of springbok, the mean cycle duration for each springbok was first calculated.
To determine the time when maximum and minimum ambient temperatures changed significantly, the month in which temperature rose above the mean +1 SD of the preceding month was marked as the onset of the increase and the month in which temperature fell below the mean -1 SD of the preceding month was marked as the onset of the decrease.
RESULTS
Springbok exhibit a mixture between continuous reproductive cycling and periods of synchronized anestrus that are not predictable from year to year. The occurrence of luteal phases expressed by the eight individual springbok ewes is depicted in Figure 1 and the annual change in the percentage of ewes expressing estrous cycles, as detected by changes in progesterone concentrations, in Figure 2. During the first year of the study, when springbok were becoming sexually mature, seven had started to exhibit estrous cycles by January 1995, which continued to June 1996, whereupon the eighth also displayed ovarian activity. All ewes became quiescent between November 1996 and March 1997 (range, 124–161 days; mean, 140 ± 5 days) and a synchronous onset in estrous cycles was noted between 25 March 1997 and 22 April 1997. The period between luteal phase onsets, which is equivalent to the length of the estrous cycle, averaged 16.4 + 0.4 days. In six ewes, cycling ceased again between 26 December 1997 and 27 January 1998 (range, 73–179 days; mean, 132 ± 15 days), but two ewes continued to cycle until the end of the study. The six anestrous ewes started to cycle again between 19 May 1998 and 5 June 1998.
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It is apparent that the fluctuations observed in circulating progesterone were associated with distinct preovulatory LH surges (Fig. 3). A surge in LH secretion was observed in all ewes 55 ± 5 h after the fall in progesterone. Progesterone levels increased again 45 ± 8 h after the surge. Photoperiodic information is transformed into a biochemical signal in the springbok ewe as there was a clear difference in the release of melatonin during the day and night (Fig. 4).
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DISCUSSION
This study provides a definitive endocrinological description of the annual reproductive cycle of the springbok, a species known to breed continuously throughout the year. Importantly, it reveals that there is a distinct anestrous period in most ewes but some individuals do not always cease ovarian activity. It is arguable, therefore, that there is an element of seasonal breeding in the springbok ewe. Like "typical" seasonal breeders, such as sheep and goats, these periods of anestrus are significantly synchronized between individuals but, in contrast, the time of year is not predictable. This suggests strongly that some proximate factor, other than photoperiod, is determining estrous cyclicity in the springbok ewe.
Our finding that most springbok exhibit an anestrous period agrees with studies on other species considered aseasonal: the addax, Addax nasomaculatus [17] and scimitar-horned oryx [18]. Although the study on addax was much shorter (late autumn to early spring), anovulatory periods of between 39 and 131 days were detected in individual animals. Some addax may have expressed periods of synchronized anestrus (possibly four of six), but the duration of the study did not allow for full analysis of this characteristic. More recently, the scimitar-horned oryx was found to have a synchronized anestrus that ranged from 35 to 95 days, but whether this anovulatory period was repeated between years was not investigated [4]. In contrast to these three antelope species, no evidence for periods of reproductive anestrus was detected during a 3-yr study on dik-dik [7], nor during a study over 1 yr on sable antelope [8]. Similarly, estrous cycles continue unabated in farmed chital deer (Axis axis) [19]. It is of interest that in the aseasonally breeding sambar deer, Cervus unicolor, there is also an anovulatory period but, like springbok, some individuals cycle continuously [20].
In sheep, genetic parameters have been shown to affect the timing of the onset of ovulatory activity after anestrus [21]. Thus, the interindividual differences that we have observed in springbok may be genetic, with some ewes able to cycle continuously, whereas others are responsive to some as yet unidentified, proximate factor. However, as seven ewes were cycling continuously at the start of the study, it is possible that all springbok are able to remain reproductively active throughout the period when anestrus may occur. This point should be viewed in context with the fact that because this first year encompassed the transition period between prepuberty and sexual maturity in most of our springbok, any pattern of breeding may not have emerged. Alternatively, it is possible that when springbok cycle continuously, some ovarian factor dampens the expression of a reproductive rhythm. For example, the rhythm of some hormone, such as prolactin, in the ewes that are continuously cycling may still be in synchrony with the release of that hormone in the quiescent ewes [22, 23]. Due to the nature of the present study, we could not investigate prolactin changes as springbok were acutely stressed prior to blood sample collection.
It is apparent that the absence of a seasonally predictive reproductive pattern in springbok is not due to the inability of this species to secrete melatonin. However, whether springbok are able to interpret the melatonin signal remains unknown. It is of interest that a similar interindividual difference was noted in sheep subjected to a constant photoperiod (12L:12D) for 4 yr, with some sheep expressing continuous estrous cycles, whereas others had periods of reproductive activity and quiescence [9]. However, those sheep with intermittent reproductive activity were not synchronized. Although the proximate factors responsible for the commencement and cessation of springbok reproductive activity are not known, our data provide some evidence about the relative importance of specific factors. Because the diet remained unchanged for the duration of the study, this could not have contributed to the observed synchrony of cycles in the springbok. It is possible in the wild that diet may affect ovarian activity as previously suggested [24]. Rainfall may have been responsible for inducing the synchronous onset of cycling in 1997, as all ewes started to cycle immediately after an exceptionally high rainfall in March. However, no rain had fallen before a similar degree of synchrony was evident in the six anestrous ewes in June 1998. Rather, our study raises the possibility that the annual change in temperature may play a critical role in initiating the onset of reproductive activity in the springbok. Specifically, both synchronous onsets in April 1997 and June 1998 were coincident with a significant decline in both the minimum and maximum ambient temperature.
There are numerous studies on domestic livestock demonstrating that high ambient temperatures depress reproductive efficiency (e.g., [25–27]). In the natural habitat of springbok, the Kalahari, the mean maximal temperature from November to March exceeds 35°C, with the average above 39.5°C in some months (source: South African Weather Bureau). Indeed, one speculation is that an important ultimate factor determining when springbok enter the breeding season is to avoid the extremely high summer temperatures in their natural habitat. There are, however, extremely few studies investigating ambient temperature as a proximate regulator of reproduction. This is not surprising because such studies are difficult to conduct in the absence of a suitable model because it is well established in sheep and goats that photoperiod is the primary proximate factor affecting ovarian activity. Responses to temperature, therefore, may be heavily masked by responses to photoperiod. Although some ovine studies [28] have reported a link between the onset of ovarian activity and lowered temperature, others have found no effect [29]. Studies on goats [30] and sheep [31] suggest that temperature may provide a fine-tuning mechanism to arrest ovarian activity at the end of the breeding season.
In the natural habitat of springbok, food availability and quality increase dramatically in response to rainfall [1]. Between 1994 and 1998 in the Kalahari, rainfall averaged 146 mm with 80% falling between November and March, although in 1994–1995, only 12 mm of rain fell. If ambient temperature is a principal proximate factor driving reproductive activity in wild springbok, then most offspring conceived in response to a drop in ambient temperature would be born during the hot-wet season, when food availability was most abundant (Fig. 5). Further long-term research will be required to establish whether temperature is a proximate factor affecting reproduction in other species inhabiting arid environments.
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Clearly, the different adaptations we proposed to explain aseasonal breeding are not upheld by the present study. Although some ewes were able to cycle continuously, the majority had a clear synchronous anestrus. How can we reconcile this finding with what is observed in the wild, when lambs are recorded throughout the year? Although it is possible that in their natural environment more ewes cycle continuously and thus conceive at any time in the year, we believe that the male rut may be critical in this regard. The rut is a period of intense territorial male behavioral activity characterized by loud vocalizing, increased boundary marking, and aggression toward other males and decreased foraging, lasting between 5 and 23 days [24]; the proximate factor or factors initiating the rut in springbok is unknown. Springbok rutting in the wild has only been observed from January to August [24], but this period coincides with the occurrence of anestrus in the present study. Following ruts in April and July, there were definite increases in the proportion of lambs to ewes in a herd [1], but whether the ewes were already cycling or anestrus at the time of the rut could not be established. During the rut in many seasonal ungulate species (e.g., impala [32]), most anestrous females come into estrus and, indeed, estrus has become synonymous with rutting during the mating season in these species. Thus, one function of the male rut may be to initiate cycling in anestrous ewes, and this could maximize the breeding capacity of springbok. Rutting has been equated to the "ram effect" in sheep, which is used to induce an earlier onset of the breeding season. Extrapolating from ovine data [33] to springbok suggests that if springbok ewes were anestrous and relied solely on the ram effect to induce ovulation, then only ruts lasting longer than 3 wk would result in maximal conception.
Combining our data with observations in the wild, where springbok clearly breed throughout the year, although with definite lambing peaks, suggests that 1) if the rut occurs during the "breeding season", the biggest increase in the lamb-to-ewe ratio occurs; 2) if the rut does not coincide with the "breeding season", then the number of females impregnated will be lower; 3) ewes that are able to cycle at any time of the year can get impregnated at other times of the year, thus giving the "appearance" that springbok breed aseasonally, which would mask any underlying seasonal effect. These ewes would be receptive during the shorter ruts; 4) during the "breeding season", when all ewes are cycling, opportunistic mating may occur outside of the rut so that some females are impregnated, including pubertal females coming into estrus for the first time. This would also give the appearance of aseasonal breeding.
In summary, our study provides novel insights into the reproductive endocrine changes occurring in a wild antelope, the springbok, which is considered to be an aseasonal breeder. Furthermore, it demonstrates that an aseasonally breeding species may express periods of anestrus, as in the addax and scimitar-horned oryx, but may also cycle continuously as in the dik-dik, which would allow for opportunistic breeding. Our study suggests that some proximate factor, possibly ambient temperature, may time the onset of estrous cyclicity in the springbok. Synchronous seasonal breeding alone is too risky in arid regions, as the ultimate reason for breeding is survival of offspring [34]. Thus, combining synchronous breeding with opportunistic breeding means that mortality rate, although probably higher, is a necessary expense for exploiting the arid niche. This gambling is essential because the springbok ewe is unable to predict climatic conditions prevailing 6 mo after conception.
ACKNOWLEDGMENTS
We thank Martin Haupt and Dominic Moss for the considerable assistance they gave in maintaining the animals and collecting blood samples, without whom this project would not have been possible. We thank the South African Weather Bureau for the meteorological data presented in this paper.
FOOTNOTES
First decision: 10 October 2000.
1 Funded by the National Research Foundation, South Africa. 
2 Correspondence. FAX: 44 117 928 9582; donal.c.skinner{at}bristol.ac.uk
Accepted: November 16, 2000.
Received: September 15, 2000.
REFERENCES
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