Effects of GnRH agonists on testosterone and testosterone-stimulated parameters for contraception and aggression reduction in male lion-tailed Macaques (Macaca silenus).

Managing social groups in zoos requires controlling reproduction in individuals that do not have a current breeding recommendation, while simultaneously maintaining social harmony and animal well-being. Contraceptives, such as gonadotropin releasing-hormone (GnRH) agonists, that suppress testosterone production, offer a potential solution. They achieve infertility by interrupting spermatogenesis and may ameliorate androgen-induced aggression. This study investigated the effects of two GnRH agonists, histrelin and deslorelin, on testosterone, testis size, body weight and sperm production in male lion-tailed macaques, along with subjective observations of aggressive behavior. Five trials at three institutions with 14 males demonstrated that 100 mg histrelin or 9 to 12 mg of deslorelin could at least temporarily reduce testosterone, but a lower 6 mg dose was ineffective. However, ability of deslorelin to produce azoospermia varied among males, even at the highest dose. In general, a higher dose was needed (1) to achieve than to maintain suppression of any measured parameter and (2) to suppress sperm production than testosterone concentration. Testosterone production was also more likely than sperm production to recover, suggesting possible damage to seminiferous tubules but not to Leydig cells. Aggressive behavior was reduced in all but the group receiving the lowest dose. This allowed social groups to be maintained for many years despite recovery of testosterone in some males, suggesting that new social roles had been learned and become independent of androgen influence.

DESLORELIN (SUPRELORIN®) USE IN NORTH AMERICAN AND EUROPEAN ZOOS AND AQUARIUMS: TAXONOMIC SCOPE, DOSING, AND EFFICACY.

The Association of Zoos and Aquariums Reproductive Management Center (RMC) in the US and the European Association of Zoos and Aquaria Reproductive Management Group (RMG) in Europe monitor efficacy of contraceptive products in participating institutions and use those results to inform contraceptive recommendations. This study used the joint RMC-RMG Contraception Database to analyze efficacy of deslorelin implants (Suprelorin®), a contraceptive used in a wide range of mammalian taxa. More recently its use has increased in birds and in some reptiles and fish. Deslorelin, a gonadotropin-releasing hormone (GnRH) agonist, stimulates the reproductive system before downregulating receptors on pituitary cells that produce hormones that stimulate gonadal steroids in both males (testosterone) and females (estradiol and progesterone), interrupting sperm production and ovulation, respectively. Nevertheless, it has been used mostly in females. Efficacy has been high in mammals, with failures resulting in offspring in only 1.3% of treated individuals and 0.5% of treatment bouts. The failure rate has been higher in birds, with 14.7% of individuals in 7.2% of bouts producing eggs, perhaps reflecting differences in avian GnRH molecules. Too few reptiles and fish have been treated for meaningful analysis. Although deslorelin appears very safe, a possible exception exists in carnivores, because the stimulatory phase can result in ovulation and subsequent sustained progesterone secretion that may cause endometrial pathology. However, the stimulatory phase can be prevented by treatment with megestrol acetate for 7 d before and 7 d after implant insertion. The two current formulations of Suprelorin are effective for minimums of 6 (4.7 mg) or 12 mo (9.4 mg). The data indicate that Suprelorin is an effective and safe contraceptive option for female mammals, although it may not be effective in males of some mammalian species. Further research is needed to ascertain its usefulness in nonmammalian taxa.

Melengestrol acetate contraceptive implant use in colobus monkeys (Colobus guereza): Patterns through time and differences in reproductive potential and live births.

Melengestrol acetate (MGA) implants are progestin-based reversible contraceptives used to manage fertility in zoo populations. Although it is recommended that MGA implants should be replaced every 2 years, the duration of efficacy has not been systematically evaluated in most species. Anecdotal reports for Old World monkeys indicate that reproduction may be suppressed longer if the implant is not removed. This study uses Guereza colobus monkey (Colobus guereza) as a model Old World monkey species to examine the effects of MGA implants on reproductive potential. In particular, we investigate whether the probability of reproducing (pR) and rates of stillbirth differ among (1) non-implanted females, (2) females who have had MGA implants removed, and (3) females whose implants were left in past expiration. We found no significant difference in pR between non-implanted and implant-removed groups, but when implants were left in past expiration, the pR was significantly lower than in other groups. Both parity and age significantly impacted pR for the non-implanted group (i.e., younger females and those who were parous increased pR), but neither were significant factors for the implant-removed group. Stillbirth rates were significantly higher post-contraception as compared with pre-contraception. These results support similar analyses in other taxa that show a shorter time to reversal after MGA contraception when implants are removed, making this a good contraceptive option for females likely to receive a breeding recommendation, especially when a more predictable time to reversal is important.

Improving the sustainability of ex situ populations with mate choice.

Many breeding programs managed by the Association of Zoos and Aquariums' (AZA) Species Survival Plans® (SSPs) are not meeting goals for population size and genetic diversity due to failure of recommended pairs to breed successfully. According to AZA Population Management Center analyses, as many as 80% of recommended breeding pairs fail to produce young before the next breeding and transfer plan is issued. Determining reasons for failure and ensuring that a specific pairing produces offspring can be challenging. Mate incompatibility, one possible reason for failure, might be addressed by allowing mate choice. Although many SSP® coordinators and breeding managers, who implement breeding recommendations at their institutions, recognize the potential benefits of mate choice, examples and models for presenting and assessing choice are lacking. Here we review examples from birds, rodents, lagomorphs, marsupials, carnivores, fishes, and insects where mate choice has been incorporated. These examples provide strong evidence that free mate choice and mating with preferred partners increase a variety of reproductive success measurements when compared to assigned mate pairings. We suggest innovative housing and breeding arrangements for better incorporating mate choice into the management strategies for species held ex situ. Further, we discuss the fitness consequences and welfare implications of allowing choice. We advocate for a more systematic use of behavioral research in cooperative breeding programs. Behavioral management for mating can yield more successful programs, thus ensuring SSP® genetic and demographic goals are met, while simultaneously improving welfare.

Reactions of female cheetahs (Acinonyx jubatus) to urine volatiles from males of varying genetic distance.

Understanding the factors used by female cheetahs (Acinonyx jubatus) to make mate choice decisions could benefit zoo breeding programs, which currently assign mates based primarily on genetic distance. Because transporting animals between institutions is costly and can be stressful, females are often limited in the number of males available for mating. One solution would be to determine if an easily transported substance could be used to gauge interest by a female to a potential mate. Here, we investigate female interest in urine samples from males of different genetic distances. Twelve females at five institutions were offered scents from 17 males of varying genetic relatedness in a pair-wise choice paradigm. Behavioral responses of the females were recorded to determine preference. Results showed that females spent more time sniffing and in proximity to scents from the most distantly related males, but female response was not influenced by male urine testosterone concentration, female parity, age, or estrous cycling. Further research will be necessary to determine whether a female's interest in male urine translates to mate preference and acceptance before this technique can be applied to zoo breeding programs.

Contraception in Dogs and Cats.

Although surgical sterilization is the most common approach to reproductive management of dogs and cats in North America and many other places, there is growing interest in nonsurgical and reversible methods. This review summarizes the methods currently available for use as reversible contraception in dogs and cats, along with cautions about reported side effects. The products covered include synthetic steroid hormones, peptide hormone agonists, vaccines, and a barrier method.

Characterization of estrous cycles and pregnancy in Somali wild asses (Equus africanus somaliensis) through fecal hormone analyses.

Although reproduction in the domestic horse has been well described, less is known about reproduction in wild equids. This study describes endocrine patterns associated with estrous cycles and pregnancy for Somali wild asses (Equus africanus somaliensis), an endangered African equid. Fecal samples were collected three times per week for more than 2 years from five female Somali wild asses at the Saint Louis Zoo; progestagen and estrogen metabolites were quantified using commercially available immunoassays. Progestagen analysis indicated that cycle lengths were 27.2 ± 1.2 days and females cycled throughout the year. Progestagen levels during early pregnancy were low and not sustained above baseline until approximately 40 weeks prior to partition. Concentrations increased markedly around 16 weeks prior to delivery and peaked 2-3 weeks before birth. Fecal estrogen levels also increased significantly starting 40-45 weeks before parturition and reached their maximal value approximately 20 weeks prior to birth. Neither foal heat nor lactational suppression of estrus was observed, and females cycled within 45 days after delivery. These data are the first to describe the reproductive physiology of Somali wild asses. As the species faces increasing threats in the wild, this information may support conservation efforts by assisting with ex situ breeding programs.

Studies of wolf x coyote hybridization via artificial insemination.

Following the production of western gray wolf (Canis lupus) x western coyote (Canis latrans) hybrids via artificial insemination (AI), the present article documents that the hybrids survived in captivity for at least 4 years and successfully bred with each other. It further reports that backcrossing one of the hybrids to a male gray wolf by AI also resulted in the birth of live pups that have survived for at least 10 months. All male hybrids (F1 and F2) produced sperm by about 10 months of age, and sperm quality of the F1 males fell within the fertile range for domestic dogs, but sperm motility and morphology, in particular, were low in F2 males at 10 months but improved in samples taken at 22 months of age. These studies are relevant to a long-standing controversy about the identity of the red wolf (Canis rufus), the existence of a proposed new species (Canis lycaon) of gray wolf, and to the role of hybridization in mammalian evolution.

Variable duration of reproductive suppression in male coyotes (Canis latrans) treated with a high dose of the gonadotrophin-releasing hormone agonist deslorelin.

Effective and humane management strategies for coyotes (Canis latrans) remain elusive. We hypothesised that exposure to a high dose of a gonadotrophin-releasing hormone (GnRH) agonist would cause prolonged suppression of the reproductive axis. Two groups of male coyotes were administered 47mg deslorelin in the form of either five 9.4-mg controlled-release Suprelorin (Peptech Animal Health, Macquarie Park NSW, Australia) implants (n=3) or 10 4.7-mg implants (n=5). In the first group, deslorelin suppressed plasma LH, testosterone and testes volume in two of three coyotes for three breeding seasons. In the second group, two of five deslorelin-treated coyotes had no sperm production after 1 year and plasma LH, FSH, testosterone and testes volume were suppressed. Although plasma gonadotropins and testosterone were suppressed in three treated coyotes in group two, testes volume and sperm production were evident. Because the duration of suppression differed among individual coyotes, we further hypothesised that a variation in deslorelin release underlay the variability. To test this, we analysed in vivo plasma profiles of deslorelin concentrations. These profiles suggested that deslorelin concentrations >100 pg mL-1 are required to maintain suppression in male coyotes. For field implementation, the development of an implant capable of releasing deslorelin for the life of the coyote is necessary.

A survey of bonobo (Pan paniscus) oral contraceptive pill use in North American zoos.

Contraception is an essential tool in reproductive management of captive species. The Association of Zoos and Aquariums (AZA) Reproductive Management Center (RMC) gathers data on contraception use and provides recommendations. Although apes have been given oral contraceptive pills (OCPs) for at least 30 years, there have been no published reports with basic information on why the pill is administered, formulations and brands used, and effects on physiology and behavior. Here, we report survey results characterizing OCP use in bonobos (Pan paniscus) housed in North American zoos, as well as information accumulated in the RMC's Contraception Database. Of 26 females treated, there have been no failures and nine reversals. The most commonly administered OCP formulation in bonobos contained ethinyl estradiol (EE) 35 µg/norethindrone 1 mg. Few females on combined oral contraceptives (COCs) were given a continuous active pill regimen; a hormone-free interval of at least 5 days was allowed in most. Crushing the pill and mixing with juice or food was common. Females on COCs seldom experienced breakthrough estrus or bleeding, while these conditions were sometimes observed for females on continuous COCs. All females on COCs exhibited some degree of perineal swelling, with a mean score of 3 or 3+ most commonly reported. Behavioral changes included less sexual behavior, dominant females becoming subordinate, and a negative effect on mood. No appreciable change in weight was noted. Taken together, these results indicate that OCPs are an effective and reversible contraceptive option for bonobos that can be used by zoos and sanctuaries to limit reproduction. Zoo Biol. 35:444-453, 2016. © Wiley Periodicals, Inc.

Parent-offspring behavior of Jambu fruit doves (Ptilinopus jambu).

Fruit doves (Ptilinopus) constitute a genus of small to medium-sized, brightly colored arboreal birds, whose diets consist entirely of fruit. Little is known about the behavior of fruit doves because most species inhabit dense forests and are difficult to observe in the wild. This study describes the parental behavior of Jambu fruit dove pairs (Ptilinopus jambu) in a captive breeding program at the Saint Louis Zoo. Continuous video recordings were made of three pairs which raised a total of eight squabs over 2 years; daily rates of parental and squab behaviors were quantified. Overall, females were present at the nest, brooded their squabs, pecked, and attempted feedings more often than males. Parents also cared for their squabs at different times throughout the day. Males fed and brooded squabs during the middle of the day, while females fed throughout the day and brooded in the morning, evening, and overnight. Feeding rates were lower than those described for seed-eating doves (Columbinae), with hours between consecutive feedings, and squabs rarely begged before feeding events. Most squab behaviors involved initiating or terminating brooding and self-preening. These behaviors increased as squabs approached fledging, and coincided with a shift from full to partial brooding, and a decrease in parental allopreening. Older squabs also initiated feeding less frequently. Together, these data provide the first description of parental behavior in a Ptilinopus fruit dove. The results of this study may help improve captive breeding efforts, which are likely to become increasingly important for future conservation and reintroduction programs.

Characterization of Ovarian Steroid Patterns in Female African Lions (Panthera leo), and the Effects of Contraception on Reproductive Function.

Because of poor reproduction after the lifting of an 8-year breeding moratorium, a biomedical survey of female lions in U.S. zoos was initiated in 2007. Fecal estrogen (FEM), progestagen (FPM) and glucocorticoid (FGM) metabolites were analyzed in samples collected 3-4 times per wk from 28 lions at 17 facilities (0.9-13.8 yr of age) for 4 mo-3.5 yr and body weights were obtained ~monthly from 17 animals at eight facilities (0.0-3.0 yr of age). Based on FEM, estrous cycle length averaged 17.5 ± 0.4 d in duration, with estrus lasting 4.4 ± 0.2 d. All but one female exhibited waves of estrogenic activity indicative of follicular activity; however, not all females expressed estrous behaviors (73%), suggesting silent estrus was common. Female lions experienced puberty earlier than expected; waves of estrogenic activity were observed as young as 1.1 yr of age, which may be related to a faster growth rate of captive vs. wild lions. Mean gestation length was 109.5 ± 1.0 d, whereas the non-pregnant luteal phase was less than half (46.0 ± 1.2 d). Non-mating induced increases in FPM were observed in 33% of females housed without a male, consistent with spontaneous ovulation. A number of study animals had been contracepted, and the return to cyclicity after treatment withdrawal, while variable, was ~4.0 yr and longer than the 1-yr expected efficacy, especially for those implanted with Suprelorin. For FGM, there were no differences in overall, baseline or peak mean concentrations among the age groups or across seasons, nor were there any relationships between reproductive parameters and FGM concentrations. Overall, results suggest that poor reproduction in lions after the breeding moratorium was not related to altered adrenal or ovarian steroid activity, but for some females may have been a consequence of individual institutions' management decisions.

Reproductive behavior of the great hornbill (Buceros bicornis).

Great hornbills (Buceros bicornis) are a long-lived, monogamous species that forms strong pair-bonds, and mate compatibility is thought to be important for successful reproduction. Within AZA, great hornbills are listed as a red SSP. The population consists of a limited number of individuals that do not breed reliably, and improving reproduction is a top priority for the Coraciiformes TAG. To better understand mating behavior and evaluate mate compatibility, this study documented the behavior of pairs of great hornbills during and immediately after courtship. Using live observations, the study followed one female, an experienced and successful breeder, as she was paired with four successive males over 11 breeding seasons. Initially, males frequently vocalized, investigated the nest, and approached the female. As the female spent more time in the nest, these behaviors were replaced by regurgitation and food offering. The female was most often observed plastering and vocalizing. Behavioral differences between successful and unsuccessful pairs, possibly indicative of pair compatibility, included rates of approaching, billing, and biting. Numerous behaviors occurred more frequently during years that a chick hatched, including pseudoregurgitation, regurgitation, offering food items, and nest investigation. Males also spent more time in proximity to both the female and the nest during years that a chick hatched. Together, these results suggest that the amount of time pairs spend in proximity, the amount of time a male spends near the nest, and the frequency of certain behaviors may help evaluate compatibility and the likelihood of successful reproduction for pairs of great hornbills.

Production of hybrids between western gray wolves and western coyotes.

Using artificial insemination we attempted to produce hybrids between captive, male, western, gray wolves (Canis lupus) and female, western coyotes (Canis latrans) to determine whether their gametes would be compatible and the coyotes could produce and nurture offspring. The results contribute new information to an ongoing controversy over whether the eastern wolf (Canis lycaon) is a valid unique species that could be subject to the U. S. Endangered Species Act. Attempts with transcervically deposited wolf semen into nine coyotes over two breeding seasons yielded three coyote pregnancies. One coyote ate her pups, another produced a resorbed fetus and a dead fetus by C-section, and the third produced seven hybrids, six of which survived. These results show that, although it might be unlikely for male western wolves to successfully produce offspring with female western coyotes under natural conditions, western-gray-wolf sperm are compatible with western-coyote ova and that at least one coyote could produce and nurture hybrid offspring. This finding in turn demonstrates that gamete incompatibility would not have prevented western, gray wolves from inseminating western coyotes and thus producing hybrids with coyote mtDNA, a claim that counters the view that the eastern wolf is a separate species. However, some of the difficulties experienced by the other inseminated coyotes tend to temper that finding and suggest that more experimentation is needed, including determining the behavioral and physical compatibility of western gray wolves copulating with western coyotes. Thus although our study adds new information to the controversy, it does not settle it. Further study is needed to determine whether the putative Canis lycaon is indeed a unique species.

Factors associated with uterine endometrial hyperplasia and pyometra in wild canids: implications for fertility.

The ability to safely and effectively manage reproduction is central to the success of AZA captive-breeding programs. Although the AZA Wildlife Contraception Center routinely monitors contraceptive safety, there have been no studies that compare the effects of contraceptive use to separation of males from females, the other option for preventing reproduction. We used retrospective medical records and pathology reports submitted by AZA and related facilities for the seven AZA-managed canid species to assess rates of uterine pathology relative to female reproductive life histories. Our results showed that the prevalence of both pyometra and endometrial hyperplasia (EH) was associated not only with treatment with the two most common contraceptives (Suprelorin® and MGA implants) but also with the number of years barren (i.e., not producing a litter and not contracepted). Rates of pyometra and EH were especially high in African painted dogs and red wolves, but lowest in swift and fennec foxes. The number of years producing a litter had a low association, suggesting it could be protective against uterine pathology. A more recently developed Suprelorin® protocol using Ovaban® to prevent the initial stimulation phase, followed by implant removal when reversal is desired, may be a safer contraceptive option. These results concerning the relationship between reproductive management and uterine health have important implications for AZA-managed programs, since the unsustainability of many captive populations may be due at least in part to infertility. Managing a female's reproductive lifespan to optimize or maintain fertility will require a reconsideration of how breeding recommendations are formulated.

"Use it or lose it": characterization, implications, and mitigation of female infertility in captive wildlife.

Zoos and other ex situ wildlife institutions can play an important role in species conservation by maintaining populations for education and research, as sources for potential re-introduction or reinforcement, and as ambassadors for financial support of in situ conservation. However, many regional zoo associations are realizing that current captive populations are unsustainable, with many programs failing to meet demographic and genetic goals to ensure long-term viability. Constraints on population size due to limited space often mandate delayed and/or less frequent breeding, but for females of many species this can have profound effects on fertility. A retrospective analysis combined with published literature and reliable anecdotal reports reveals that, when females are housed in a non-breeding situation for extended periods of time, reproductive changes that negatively impact fertility have occurred in multiple species, including canids, elephants, white rhinoceros, Seba's bats, wildebeest, stingrays, and some felid species. Competing space needs and changing interest in taxa for exhibits over time compound the problem. Counter strategies to breed early and often have their own demographic and genetic consequences as well as logistical and political implications. Strategies to mitigate the sustainability crisis in these taxa might include a mixed strategy in which young, genetically valuable females are bred earlier and at more regular intervals to ensure reproductive success, in combination with the judicious use of available tools to manage the number of offspring produced, including contraception and culling. An understanding of the issues at stake is the first step towards developing management strategies for sustainable populations.

Kinematic activity of gray wolf (Canis lupus) sperm in different extenders, added before or after centrifugation.

We evaluated two approaches to improving in vitro wolf sperm survival. Both approaches aimed to reduce the exposure of sperm to prostatic fluid resulting from electroejaculation: (1) use of extender formulations recently developed for the domestic dog (the most closely related domestic species); and (2) dilution of ejaculate shortly after semen collection. Three commercial extenders were compared with the TRIS-based extender we had previously used. We also compared the effects on motility of adding extender immediately after collection to our previous protocol in which extender was added after centrifugation. Both subjective and objective (computer-assisted semen analysis program) kinematic measurements were made. Relatively minor differences were noted (and not in total or progressive motility) between the centrifugation protocols. Two of the commercial extenders resulted in significant improvement in motility over the TRIS-based extender and one of the other commercial extenders at 8 hours after collection (mean ± SEM; total motility was 68.3 ± 4.0% and 70.0 ± 4.0% compared with 53.3 ± 4.0% and 55.0 ± 4.0%, respectively; progressive motility 58.6 ± 5.4% and 57.1 ± 5.4% compared with 32.8 ± 5.4% and 39.3 ± 5.4%; P < 0.05). We inferred that components in two of the commercial dog extenders might provide more protection for wolf sperm, prolonging their motility.

Affiliative and aggressive behavior in a group of female Somali wild ass (Equus africanus somalicus).

We observed a group of three young female Somali wild asses to develop an ethogram of social behavior in the first phase of a longer term study of social, sexual, and maternal/infant behavior. The most unexpected finding was the frequency and extent of aggressive interactions, which included Charge, Drive, Neck Wrestle, Head Butt, and Body Slam, behaviors previously reported only for males of other equid species. The overall frequency of aggressive behavior was higher than that of affiliative behavior (84±16.5 vs. 32±5.5, P=0.03), yet no injuries occurred. The dyadic directionality of aggressive behavior suggested a dominance hierarchy, a feature not previously reported for either wild ass or domestic donkeys. The aggression observed may be an accurate representation of the behavior of this species, or their relatively young ages, or their recent transfer from their natal group through quarantine and into a new enclosure may have heightened agonistic tendencies. Further studies will determine whether with time their aggressive behavior becomes more intense or dissipates with maturity.

Effect of semen collection method on sperm motility of gray wolves (Canis lupus) and domestic dogs (C. l. familiaris).

Genetic management of Mexican gray wolves includes semen banking, but due to the small number of animals in the population and handling restrictions, improvements in semen collection and cryopreservation rely on results from studies of domestic dogs. Semen collection from wolves requires anesthesia and electroejaculation, which introduce potentially important variables into species comparisons, as dog semen is typically collected manually from conscious animals. To investigate possible effects of collection method on semen quality, we compared semen collection by the traditional manual method and by electroejaculation (EE) in a group of dogs (n = 5) to collection by EE only in wolves (n = 7). Samples were divided into two aliquots: neat or diluted in Tris/egg yolk extender, with motility evaluated at intervals up to 24 h. There were no differences (P > 0.10) in sperm motility in either neat or extended samples at 24 h from EE dogs and wolves, although motility of the wolf neat samples declined more rapidly (P < 0.05). However, there were differences (P < 0.01) between EE and manually collected dog semen in motility at 24 h, in both the neat and extended samples. Therefore, general motility patterns of dog and wolf semen collected by EE were similar, especially when diluted with a Tris/egg yolk extender, but sperm collected from dogs by EE did not maintain motility as long as manually collected samples, perhaps related to the longer exposure of EE samples to more prostate fluid.

Nutrition and behavior of fennec foxes (Vulpes zerda).

Fennec foxes make popular pets because of their small size, minimal odor, and highly social behaviors. They are kept in zoos for conservation and educational programs. The exotic animal practitioner is most likely to be presented with fennec foxes that are overweight because of inappropriate diets or excessive feeding. Clients attempting to hand-rear fennec foxes need advice about formula selection, amounts to feed, protocols for keeping pups warm, and weaning. This article provides information on social behavior, reproduction, and parental behavior, nutrition, and hand-rearing.