Use of a custom restraining box for estrus induction using PMSG and successful reproduction in a snow leopard (Panthera uncia): a case report

Abstract

Importance

Snow leopards (Panthera uncia) are vulnerable felids with low reproductive success in captivity. Their thick fur and cautious behavior often make dart-based hormone delivery unreliable; as a result, practical estrus-induction protocols are limited.

Case Presentation

A 9-year-old female snow leopard at Seoul Zoo, housed with a male since 2003 without previous conception, underwent two estrus induction attempts. In the first trial, 600 IU pregnant mare serum gonadotropin (PMSG) followed by 300 IU human chorionic gonadotropin resulted in mating but only a pseudopregnancy. A portable wooden restraining box with a squeezing panel enabled accurate hormone injection using hand syringes. After a 97-day separation period, a second trial involved a single injection of 600 IU PMSG. After reintroduction to the male, multiple matings occurred for eight days, and a single cub was delivered 94 days after the first mating. The cub nursed normally for two days but died due to maternal infanticide.

Conclusions and Relevance

This case shows that reliable estrus induction and parturition in snow leopards can be achieved with a single PMSG injection when supported by a customized restraining box that ensures accurate hormone delivery. This method offers a practical approach to assisted reproduction in difficult-to-handle snow leopards and other felids.

INTRODUCTION

The snow leopard (Panthera uncia) is classified as “vulnerable” on the International Union for Conservation of Nature and Natural Resources Red List. Among nondomestic felids in captivity, the snow leopard is among the most difficult to breed, either naturally or through exogenous hormone-induced estrus [1,2,3,4,5,6]. A major reason for the limited breeding success is that pregnancy rarely occurs despite repeated behavioral estrus and multiple coitus observed in zoos [1,5]. Various doses of exogenous hormones have been used to improve the success rate of captive breeding in wild felids, depending on species-specific ovarian responsiveness [2,3,4,5].

Unlike other large cats, snow leopards have extremely thick and fine fur adapted to the severe, long winters of their high-altitude habitats in Central and South Asia. This dense coat complicates hormone administration. Although CO₂-powered dart guns or blow darts are used widely to deliver anesthetics or hormones to zoo or wild animals [7,8,9,10], the expected dose may not be fully delivered in snow leopards because the lateral opening of the needle can discharge prematurely within the dense fur before reaching the skin. Moreover, installing a restraint device at every enclosure is challenging in zoo settings. Therefore, a portable restraining box capable of luring and securely holding the animal is necessary for accurate hormone injection with hand syringes and for other medical procedures.

A pair of snow leopards—stud number 2,324 (female) and 2,290 (male)—had been housed together in the same exhibition enclosure at Seoul Zoo since their introduction from Germany and France, respectively, in 2003. Despite continuous cohabitation, no pregnancy had occurred, whereas tigers and lions housed heterosexually at the zoo often exhibited overbreeding. This retrospective study describes two breeding attempts conducted from September 2011 to October 2011, using exogenous hormones administered via a handmade wooden restraining box to induce estrus and produce offspring in captive snow leopards.

CASE PRESENTATION

This study was conducted with the approval of the Seoul Zoo Executive Council, which served as the ethical oversight body before the establishment of an Institutional Animal Care and Use Committee. A female snow leopard born in June 2002 at Tierpark Berlin Zoo, Germany, and a male snow leopard born in May 2001 at Mulhouse Zoo, France, were introduced to Seoul Zoo in May and August of 2003, respectively. At the time of hormone administration, the body weight of the female snow leopard was 38 kg. Since then, the pair had never been separated and lived together in the same enclosure.

Before beginning the study, a rectangular cuboid plywood box (150 × 80 × 120 cm; length × width × height) was handmade (Fig. 1A). All 12 edges were reinforced with steel rack rails. One longitudinal side of the box contained circular holes for hormone injection, while the opposite side had two holes connected to metal bars used to operate an internal squeezing panel. The top surface was fitted with a dome-shaped transparent plastic cap, allowing visual monitoring of the female until she adopted a suitable posture for restraint. Chicken meat was placed deep inside the box as a lure. When the female stepped on the foot panel positioned before the bait, a rope-connected sliding backdoor automatically closed (Fig. 1B and C).

Fig. 1. Portable restraining box and procedure for accurate hormone administration in a female snow leopard. (A) The female snow leopard hesitates at the entrance of the wooden restraining box designed for a safe manual injection. (B) Upon stepping on the internal foot panel, the sliding backdoor automatically closes, securing the animal inside the box. (C) The female becomes fully restrained in the box, allowing controlled positioning using the internal squeezing panel. (D) Through a lateral injection port, the exact volume of hormone is administered intramuscularly using a hand-held syringe.

On October 13, 2011, before the pair was separated, the female snow leopard was injected with 600 IU of pregnant mare serum gonadotropin (PMSG, Folligon^®; Intervet, Netherlands), followed 80 h later by 300 IU of human chorionic gonadotropin (hCG, Chorulon^®; Intervet) (Fig. 1D). On November 3, 2011, the female was moved to another enclosure located approximately 200 m away from the original site where she had been housed with the male since 2003.

After the expected parturition date had passed without birth, a second hormonal trial was conducted. On February 8, 2012, the female received a single injection of 600 IU PMSG. The next day (February 9), she was returned to the male’s enclosure, and mating was observed from February 10 to February 17. On April 4, the female was transferred again to the secluded enclosure where she had previously been separated. On May 14, 2012, a single cub was born at approximately 7:00 P.M. (Fig. 2A and B), 94 days after the first mating. The mother nursed well (Fig. 2C), but early on May 16, the two-day-old cub was consumed by its mother.

Fig. 2. Parturition and early postpartum behavior following estrus induction with a single PMSG injection. (A) A single snow leopard cub was delivered spontaneously on May 14, 2012. (B) The dam immediately licks and stimulates the newborn cub after parturition. (C) The dam and cub rest together in the secluded maternity enclosure shortly after birth. Arrowheads indicate the newborn cub.

PMSG, pregnant mare serum gonadotropin.

DISCUSSION

At Seoul Zoo, a pair of snow leopards successfully produced two litters in 1989 and 1991. Despite this success, after a new pair was introduced in 2003, no cubs had been produced for many years. To ensure that the full volume of hormones, antibiotics, or vaccines is administered, zoo animals typically require specialized restraint devices or anesthetics to limit movement, or at least some method to slow them down [11,12]. In many cases, however, especially with highly sensitive species, their sharp instincts and ability to detect potential threats prevent them from approaching within the effective range for darting, whether with blowguns or rifles [7,8,9]. This is particularly true for snow leopards, whose thick fur, cautious nature, and rapid movement make accurate hormone delivery difficult. Hormones can be splattered or lost when the lateral opening of the needle is exposed prematurely due to the dense fur, even if the animal remains still before darted. This issue has also been observed in other species with thick coats or in animals that do not allow perpendicular injections because of constant or sudden movement.

For these reasons, a restraining box was used in this study. The restraining box was equipped with transparent half-sphere viewing domes and round injection holes. The box was highly effective, allowing precise hormone delivery using hand syringes while the snow leopard remained immobilized with the internal squeezing panel (Fig. 1). At Seoul Zoo, this approach is considered a pilot reproductive management strategy and is applied cautiously on a case-by-case basis, with careful consideration of individual animal conditions, seasonality, and animal welfare. In contrast to the first trial, when the female readily entered the box twice, she became suspicious during the second trial and could not be lured in for the scheduled hCG injection 80 h after PMSG. The inability to administer hCG in both trials may be considered a limitation of the protocol, as no alternative ovulation induction strategy was implemented when box luring failed. Future protocols may benefit from incorporating a contingency approach for ovulation induction under such circumstances. Forcing her into the box was avoided because it might adversely affect follicular development [13]. Compared with the timid female, the male displayed a more curious and active personality, making it easier to lure and restrain him with the sliding door multiple times per day [14].

In the wild, snow leopards typically mate in late winter. The previous parturition dates at Seoul Zoo were May 21, 1989, and June 10, 1991 (37°21′ E, 126°59′ N), similar to the breeding patterns reported in zoos at comparable latitudes, where mating usually occurs from mid to late February. At Seoul Zoo, however, the snow leopards demonstrated year-round mating behavior (brief mounting, short receptivity, and low mating frequency) without resulting pregnancies.

In the first trial (October 2011), PMSG and hCG were administered on October 13 and October 16, respectively, and mating continued until October 22. After the female was moved to the secluded enclosure, “calling,” a typical estrus behavior, was observed beginning January 4, 2012. Snow leopards are seasonal breeders and induced ovulators [1,5,15]. They show ovarian responsiveness to exogenous hormones year-round, with follicles and corpora lutea developing 45–50 h after hCG administration [5]. In this study, the pseudopregnancy period lasted 78 days from the assumed ovulation date (October 18), which is consistent with previously reported nonpregnant luteal phases ranging from 11 to 72 days [15]. The hCG administered likely induced ovulation in this case. Male fertility may also have been insufficient outside the peak mating season to achieve pregnancy [16]. The pair exhibited sexual behavior throughout the year, but the frequency, duration, and intensity varied [17]. Previous research also suggests that females housed with males exhibit lower estrogen and progesterone levels than singly housed females, contributing to repeated pseudopregnancies [13].

From January 4 to 18, 2012, the female displayed typical “calling” behavior associated with the follicular phase. In the second trial, after 97 days of separation, the female was injected with 600 IU PMSG at 1:40 P.M. on February 8, 2012. She was returned to the male’s enclosure on February 9, and mating began at 10:00 A.M. on February 10 and continued until February 17, a period longer than the reported average of 4.3 days [15]. Although a hCG injection was scheduled for 9:40 P.M. on February 11, the female was not successfully lured into the box, and the injection could not be administered. Considering that felids are induced ovulators, the prolonged mating period of approximately eight days may have provided sufficient copulatory stimulation to trigger natural ovulation in this case. The reported estrous cycle length of snow leopards is approximately 12.7 days [15]. No estrus signs were observed before the female was transferred back to the secluded enclosure on April 4.

A single cub was born at approximately 7:00 P.M. on May 14 (Fig. 2A), 94 days after the first mating on February 10, consistent with reported gestation lengths of 93–95 days in captive snow leopards [15]. The litter size in the wild is usually two to three cubs, whereas captive litters typically contain one to two cubs, reflecting the lower pregnancy rates after natural or artificial breeding [5,15]. A single-cub litter in this case may have been influenced by the suboptimal semen quality or reduced ovarian responsiveness due to the long-term cohabitation of the pair [13,16,18]. Active maternal infanticide, as occurred in this case, is rare in the wild. Although the secluded enclosure was quiet and inaccessible to zoo visitors, maternal inexperience and environmental stress may have contributed, particularly after excessive cub-carrying behavior [19].

In conclusion, the restraining box used in this study facilitated safe animal handling and precise hormone administration and enabled the successful production of a snow leopard cub with a single PMSG injection. This is the first report of successful cub production in this species following PMSG administration. Since the successful pregnancy and parturition described in this report, no additional estrus induction or breeding attempts using the same protocol have been performed in this female. Therefore, further studies involving additional cases will be needed to evaluate the reproducibility and broader applicability of this approach. Because this report describes a single clinical case, the findings should be interpreted with caution and not generalized without further evidence from additional cases.