Abstract

Introduction

To improve breeding success, it is imperative to gain more knowledge about the reproduction biology and refine assisted reproduction technologies in species where there is a likelihood of success. The bear family (Ursidae) is an appropriate example. Some bear species, kept in few numbers in European zoos, are on the verge of extinction and efforts to conserve them are being undertaken.^4,43 Experimental research on suitable model species, which are more frequently distributed in zoos, is necessary to develop new reproduction technologies (International Panda Conference, San Diego 2000). New strategies of reproduction management based on these model species can be developed using refined methods of estrus detection, pregnancy monitoring and improved techniques for assisted reproduction. Later, these results can be transferred to target species such as the giant panda (Ailuropoda melanoleuca), Malayan sun bear (Helarctos malayanus), and spectacled bear (Tremarctos ornatus). In this study, the taxonomic relationship and the reproduction biology were examined within the Ursid family to detect suitable models for endangered target species. New approaches on reproduction management were presented and results from our recent investigations are compared with those in selected print sources.

What Do We Know About Reproduction in Bears From Literature?

In the bear family (Ursidae) there are eight species of bears in three different subfamilies (Ursinae, Tremarctinae, Ailuropodinae).¹² The status of the giant panda is widely discussed but it is believed to be an early divergent from the bear family.^3,36,49,51 Due to genetic studies on the structure of hemoglobin, the South American spectacled bear was described to be the giant panda’s closest relative and the link between the Ailuropodinae and the Ursinae. In the Ursine sub-family the polar bear (Ursus maritimus) is the closest relative of the brown bear (Ursus arctos).^11,46 Breeding between these two species has been reported.⁴⁴ The two species of black bears (Ursus americanicus, Ursus thibetanus) share a common ancestor from about 4 million yr ago but phylogenetic studies revealed that the closest relative of the American black bear is the Malayan sun bear.^12,50 The sun bear is thought to be the most ancient type of bear.^28,47 Its hemoglobin structure is the same as that of the polar bear and the American black bear.^17,18

The habitats of the brown bear and the American black bear overlap in northern America, but inter-specific breeding has not been reported from the wild.⁹ The giant panda and the Asiatic black bear have much in common concerning habitat.³³ They show a moderate ecological overlap in activity schedules and use of space but differ markedly in their feeding strategy.⁴¹ The body size of the giant panda, the Asiatic black bear, the American black bear, and the spectacled bear are similar.^12,51

The reproduction biology of most bears is very similar, although the giant panda and the Malayan sun bear are extraordinary within the Ursid family. The reproduction biology of the Ursids is characterized by two main traits—seasonality and delayed implantation.^10,19 Seasonality is largely dependent upon environmental conditions and food supply.⁵ Most bears are seasonal with a mating period ranging from spring to early summer.^15,38 The only exceptions are the Malayan sun bear and the sloth bear from the island of Sri Lanka (M. ursinus inornatus) which are both non-seasonal.^7,34 All bears, except the Malayan sun bear, have a delayed implantation.^6,33 The prolonged gestation can be more or less reduced since the implantation of the embryo is regulated by the photoperiod.² As a result, the birth of bears takes place during winter hibernation. Species without hibernation, like the giant panda, deliver in late summer, or throughout the year like the Malayan sun bear.^1,12,24 Bear cubs are always small and underdeveloped. Among all bears, giant panda cubs are the smallest and can be bottle-fed with a sun bear formula.³² The mating behavior of most bears is rather unspectacular. The giant panda is the most unusual, displaying vocalization and predominant scent marking.^{21,31,39,40,45} In the wild bears do not interbreed, but in captivity there are reports about inter-specific sexual affinities.⁶ The American black bear was described as a model species for oocyte recovery and maturation for endangered Ursids.²⁰ The structure of the placenta is very similar in the spectacled bear and the brown bear.³⁰

Recent Comparative Investigations

Morphology and Sonomorphology

To describe the sonomorphology of the male and female genital tract and to monitor the variations of the reproductive organs in detail, we performed 117 ultrasound examinations in bears of eight species.^13,23 The sonographic measurements were proven by postmortem preparations in 2.5 bears of four species. The size and weight of the sexual organs varied with the body size. Our results also revealed seasonal changes in the size of the reproductive organs (Table 1). The male giant panda was outstanding in the size of its testes, especially during breeding season, as described previously.⁸ The accessory sex glands of the giant panda most resemble those of the spectacled bear, no matter what the reproduction status. The size of the testes of the brown bear, Malayan sun bear, and sloth bear are quite similar. The size of the accessory sex glands is related to the body size.

Table 1. Testis diameters (mm) in different bear species during breeding and non-breeding seasons measured ultrasonographically

In the females, the length and weight of the reproductive tract are also related to the body size of the animals. However, the diameters of the uterus and the ovarian size vary more with the reproductive status (e.g., pregnant, pseudo-pregnant, non pregnant) than with the reproduction season.

Endocrinology

Investigations were carried out on non-invasive estrus detection and pregnancy monitoring in three bear species (giant panda, brown bear, and spectacled bear). Hormonal profiles of gonadal steroid metabolites were established at IZW from urine and feces by enzyme immunoassays (EIA).^14,29 In the giant panda, urinary estradiol (E2) was determined and used to monitor the increase in estrogens, which was usually followed by estrous behavior and reached a maximum prior to high peak receptivity. In the brown bear and the spectacled bear, several E2 peaks were measured but did not correlate with observed mating behavior. In the giant panda, a second EIA was performed to show that the baseline values of urinary gestagens followed a steady increase at the assumed time of implantation. So far in the other bear species, no urinary gestagen metabolites corresponding to the panda results have been found. In brown bears, an EIA measuring fecal gestagens made it possible to differentiate between pregnant and pseudo-pregnant females after implantation.¹³ Inclination of fecal gestagens were also described in Malayan sun bears after estrus.⁴²

In addition, seasonal changes of urinary volatile substances (volatiles) were investigated by solid-phase micro-extraction in combination with gas chromatography and mass-spectra (GCMS).²² A group of volatiles belonging to a single chemical family could be discovered in the giant panda and the brown bear. This estrogen-linked group appeared not only during the rise of E2, but also at the peak of E2. This led to the conclusion that for the increase of the estrogen-linked group of volatiles a certain E2-threshold had to be exceeded. The same group of volatiles was also found in the giant panda using a colorimetric enzyme assay. However, this enzyme assay showed several false-positive results, which could be excluded by using extraction. In the spectacled bear, the estrogen-linked group also appeared but did not correlate with E2. A second urinary volatile, a steroid-like substance, which could only be found in the giant panda, displayed increasing oscillations towards ovulation and afterwards correlated with urinary gestagen metabolites of the giant panda.

Conclusions

The collected data is still incomplete, but it contributes to basic research and a better understanding of comparative investigations in living animals. In the future, methods to predict estrus and to detect pregnancies will be much more precise. Concerning possible model species for endangered Ursids, our preliminary results led us to the following conclusions:

• Giant panda: Suitable model species for the giant panda would be the spectacled bear due to its genetic relationship and the Asiatic black bear because it occupies the same habitat and feeds on a similar diet. Each of these model species is the most equal in size and anatomy to the giant panda.
• Malayan sun bear: The Malayan sun bear is the most divergent of all the bears regarding reproduction. Due to their body size the two species of black bears are regarded as model species.
• Spectacled bear: The spectacled bear’s reproduction biology is similar to all members of the ursine subfamily, but because of its body size the two species of black bears would be more suitable model species.

Summary

The reproductive tracts of male bears (six species) and female bears (eight species) were investigated and compared in morphologic and sonomorphologic measurements. The size of the organs relied heavily on the body size of the individual and on seasonality. Female bears (three species) were tested for excretion of gonadal steroid metabolites and urinary volatile substances. A peak of an estrogen-linked group of volatiles was detected prior to estrus and pregnancies could be detected after implantation of the embryo by fecal hormone metabolites. All gained results were combined and proven by investigation of selected print sources to represent model species for endangered Ursids. For the giant panda, the Asiatic black bear and the spectacled bear were found to be suitable model species; for the Malayan sun bear and the spectacled bear, the more suitable model species would be the two black bears. It will be necessary in the future to undertake more research to close existing gaps comparing the species.

Acknowledgments

We would especially like to thank the Berlin Zoological Garden for sponsoring this study in form of a scholarship for a PhD student. Also, we are very grateful to all zoos and their staff who contributed in terms of making examinations possible, who collected samples or sent us material for postmortem analyses. Special thanks to Dr. Lydia Kolter, Zoological Garden Cologne, for constructive comments.

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