Abstract

Background

The development of reliable methods for monitoring the reproductive cycle of bears is needed to optimize breeding management in captivity. Although there is a large database describing patterns of reproductive life history, anatomy of the female genital tract and endocrinology of bears, there is little basic information about reproductive physiology.² Several species of ursids are frequently kept in zoos. Zoos and private reserves have become increasingly proactive in their management, establishing conditions necessary for successful reproduction in captivity. Increasing knowledge of reproductive behavior and animal husbandry in particular has greatly enhanced the breeding success, thus creating self-sustaining populations of some ursids in captivity, particularly brown bears (Ursus arctos arctos). The use of reproductive techniques for conservation and management programs for this species includes the introduction of effective methods of contraception. There is good evidence that bears produce progesterone during the phase of delayed implantation and we suggest that antigestagens can probably be used for prevention of implantation of the early blastocyst. The aim of the present study was to develop noninvasive procedures for monitoring the reproductive status of brown bears using transrectal ultrasonography and endocrinology to get basic data for future anticonceptive treatments during diapause.

Ultrasonography

Ultrasonographic imaging techniques have proved valuable in reproductive and veterinary studies in a wide range of exotic species. Transabdominal ultrasonography is a viable method for pregnancy detection in brown bears (Ursus arctos yesoensis).⁴ However, transabdominal ultrasound has not yet been successfully used for the visualization of the nongravid uterus, gonads and early pregnancies in ursids. The ability to assess the reproductive status of bears has been quite limited. Transrectal ultrasonographic imaging provides a noninvasive tool for observing reproductive events in wild carnivores. Some of the difficulties in viewing the entire female genital tract and monitoring structural changes of the uterus and ovaries were attributed to difficulties in positioning a miniaturized high-frequency probe into the rectum of bears. Five female captive brown bears (Ursus arctos arctos) were examined ultrasonographically twice, once in late September and once in early December, after mating had been observed during the breeding season. All animals were immobilized with a dart gun using etorphine (1.3±0.2 mg/100 kg body weight) and acepromazine (6.9±0.4 mg/100 kg body weight; Large Animal Immobilon®, C-Vet Ltd., Bury St. Edmunds) with the addition of 300 IU hyaluronidase (Hylase®, Impfstoffwerk Dessau). The anesthesia was antagonized with diprenorphine (4.1±0.3 mg/100 kg body weight; Revivon®, C-Vet Ltd., Bury St. Edmunds). The genital tracts of the immobilized females were examined by transrectal ultrasonography in lateral recumbency. Feces were removed digitally using ultrasound gel (Aquasonic 100®, Parker Laboratories Inc., Orange, NJ 07050 USA) for lubrication and the rectum was then irrigated with lukewarm water. A real-time, B-mode ultrasound scanning system (CS 9100 Oculus, Picker International GmbH, Espelkamp, D-32339, Germany) equipped with a 7.5-MHz curved linear intraoperative transducer (EUP-F 334) was used. The probe was fitted in an adapter (Ultraschallkopftraeger FT2, A. Schnorrenberg Chirurgiemechanik, Woltersdorf, D-15569, Germany) and then introduced into the rectum using ultrasound gel for coupling. The urogenital tract was scanned longitudinally from caudad to craniad. The ultrasound system described allowed successful imaging of the urogenital tract of all animals investigated. All parts of the normal female reproductive tract and of the urinary system, except the ureters, could be visualized transrectally. Verification of the structures identified sonographically in situ was accomplished by performing ex situ ultrasonographic examinations of isolated urogenital tracts in two other female brown bears postmortem. In early December, three brown bears were identified as pregnant. They were characterized by local uterine enlargement (threefold), anechoic fetal fluid and endometrial proliferation at the implantation sites. The placenta appeared as a hypoechoic, discoid structure. Echogenic fetuses were easily visible within the anechoic fluid. Head, thorax, abdomen and the extremities of the fetus were clearly distinguishable at this stage. The fetal crown-rump length was 24±4 mm and the fetal heartbeat was detectable. With careful searching over the uterine horns, the number of fetuses (1–3 fetuses/animal) and mature corpora lutea on the ovaries could be detected.

Endocrinology

Plasma concentration of progesterone and gestagens in feces were determined simultaneously. Blood samples of two nonpregnant and two pregnant animals were taken during each ultrasound investigation. Fecal samples were collected weekly from September to November. For the determination of progesterone, 0.1 ml of plasma was extracted with 2 ml petrol ether, concentrated to dryness and the residue was reconstituted in 1 ml 40% methanol. Progesterone was measured with an enzyme immunoassay (EIA) as described and validated earlier.³ The antibody used was raised against progesterone-7-carboxyethylthioether-BSA and the label was progesterone-3-carboxymethyloxime-horse radish peroxidase. Fecal samples (0.5 g) were combined with 4.5 ml methanol, agitated for 30 min. and centrifuged (15 min, 1,000 g). One-half milliliter of the supernatant was diluted with 0.5 ml water. After further dilution, progesterone equivalents were quantified using the same EIA as for plasma. The plasma concentrations of progesterone were 1 to 5.5 ng/ml in all brown bears sampled in September and December, indicating functional corpora lutea. According to the limited data available,¹ there appear to be no fundamental differences between nonpregnant and pregnant animals before and after implantation (Table 1). The preliminary results show that fecal samples obtained from 10 different female brown bears in the nonbreeding season contain low concentrations of progesterone equivalents, about 10 ng/g, whereas in fecal samples from animals with active corpora lutea, estimated concentrations of progesterone equivalents were 50–800 ng/g. A slightly increased excretion was recorded in the first two pregnant bears investigated.

Table 1. Concentration of progesterone in blood plasma (ng/ml) and progesterone equivalents in feces (ng/g) of brown bears measured by enzyme immunoassay

Fecal samples in nonbreeding season originated from 10 different animals; all other samples were collected from two individuals.

Conclusions

Transrectal ultrasonography provides a tool for efficient assessment of the reproductive status in brown bears. In contrast with transcutaneous ultrasound, the advantages of the rectal method include imaging of the ovaries, including follicles and corpora lutea, and the ability to detect the nongravid uterus and early pregnancies. Noninvasive gestagen monitoring in feces may be used for pregnancy diagnosis after implantation, an important aspect particularly for breeding management of captive bears.

Literature Cited

1.  Foresman, K.R. and J.C. Daniel. 1983. Plasma progesterone concentrations in pregnant and nonpregnant black bears(Ursus americanus). Journal of Reproduction and Fertility. 68:235–239.

2.  Johnston, L.A. and S.L. Monfort. 1994. New methods and development in bear reproductive biology. In: Proceeding of International Conference on Aspects of Bear Conservation. Uludag University Press, Bursa, Pp. 209–218.

3.  Prakash, B.S., H.H.D. Meyer, E. Schallenberger and D.F.M. va de Wiel. 1987. Development of a sensitive enzyme immunoassay (EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique. Journal of Steroid Biochemistry. 28:623–627.

4.  Tsubota, T. 1990. Studies on reproductive physiology of Hokkaido brown bear, Ursus arctos yesoensis. Japanese Journal of Animal Reproduction. 36:1–10.