Janine L. Brown

Reproductive and genetic consequences of founding isolated lion populations

Species survival is critically dependent on reproductive performance, a complex physiological process under rigorous genetic control. Classical studies of inbreeding in laboratory animals and livestock have shown that increased homozygosity can adversely affect spermatogenesis, ovulation and perinatal mortality and morbidity1–3. For wild populations, the consequences of inbreeding depression have not been examined intensively, although our recent studies of the African cheetah revealed a striking degree of genetic uniformity4,5 combined with an extremely high incidence of structurally abnormal spermatozoa (>70%) in captive6 as well as free-ranging7 males. In this study, we report definitive evidence that the reproductive function of free-ranging mammals can be impaired as a result of demographic contraction followed by inbreeding. In an examination of three distinct lion populations (two from the Serengeti ecosystem in East Africa and a third descended from lions in the Gir Forest of western India), a direct correlation was observed between genetic variability and two physiological traits, incidence of abnormal sperm and circulating testosterone, a critical hormone for spermatogenesis.

Behavioral and physiological correlates of stress in laboratory cats

Abstract Sixteen domestic cats were used to investigate the pituitary-adrenal, pituitary-gonadal and behavioral consequences of an unpredictable handling and husbandry routine. After a 10-day baseline period of standard laboratory procedures, eight cats (‘stressed cats’, STR) were subjected to a 21-day period of altered caretaking characterized by irregular feeding and cleaning times, absence of talking and petting by humans, and daily unpredictable manipulations. Eight control cats (CON group) were maintained for 21 days on the standard caretaking schedule. Behavior was recorded on time-lapse video 24 h day −1 , urine was collected daily for cortisol analyses, and hormone stimulation tests with synthetic adrenocorticotropic hormone (ACTH) and luteinizing hormone-releasing hormone (LHRH) were conducted before and after the 3-week treatment period. Results indicate that the STR cats were chronically stressed by the altered caretaking routine. Urinary cortisol concentrations were consistently elevated throughout the 3-week period, adrenal sensitivity to ACTH was enhanced and pituitary sensitivity to LHRH was reduced. Active exploratory and play behavior was suppressed, and STR cats spent more time awake/alert and attempting to hide. Hiding was negatively correlated with cortisol concentration and, therefore, may be an important behavior for coping with uncontrollable and unpredictable captive environments. These results indicate that qualitatively poor caretaking is a potent psychological stressor for confined cats that may eventually compromise reproduction through behavioral or physiological mechanisms. To promote well-being, caged cats should be provided with appropriate places for concealment.

Cortisol metabolism in the domestic cat and implications for non‐invasive monitoring of adrenocortical function in endangered felids

Three domestic cats were given i.m. injections of 3H-cortisol to determine the time course and relative proportion of excreted 3H-cortisol metabolites into urine and feces. Most urinary radioactivity was detected in the first sample collected at 3.9 ± 2.5 hr postinjection and accounted for 13.9 ± 2.1% of the total radioactivity recovered. High performance liquid chromatography (HPLC) detected four urinary metabolites, one of which (13.7% urinary radioactivity) eluted with the 3H-cortisol reference tracer and was quantifiable using a commercial cortisol radioimmunoassay (RIA). The majority of cortisol metabolites in feces (85.9 ± 2.1%) was excreted at 22.3 ± 6.2 hr. HPLC analysis detected several fecal metabolites consisting primarily of nonhydolyzable water-soluble forms, none of which eluted with 3H-cortisol or 3H-corticosterone reference tracers. No immunoreactivity was detected in HPLC-separated fecal eluates using the cortisol RIA; however, two of the more polar metabolites were quantifiable using a commerical cortisosterone RIA. The physiological relevance of the immunoreactive fecal metabolites was determined in four domestic cats given an adrenocorticotropin (ACTH) challenge. Increased serum cortisol concentrations were detected within 30 min of ACTH injection, which was maintained for at least 6 hr. A corresponding increase in fecal cortisol metabolite concentrations (ranging from 238% to 826% over individual baseline values) was observed 24–48 hr later. These data indicate that adrenocortical activity can be monitored nonivasively in the cat by measuring cortisol metabolites excreted in feces. This procedure is a potentially valuable tool for endangered felid management to help evaluate responses to physiological and psychological stressors associated with environmental conditions and husbandry practices. (This article is a US Government work and, as such, is in the public domain in the United States of America.)

Reproductive endocrine monitoring of elephants: An essential tool for assisting captive management

Considerable information now is available about the basic reproductive biology of elephants, especially females. However, as important as this knowledge is, it no longer is enough to simply compile it into a database. The potential exists for using endocrine monitoring techniques to solve real problems. This review summarizes our current knowledge of elephant endocrinology and offers suggestions on how to use the technology to maximize reproductive potential. The estrous cycle can be monitored through the analysis of serum progestogens, primarily 5α-reduced compounds, and consists of an 8- to 12-week luteal phase and a 4- to 6-week inter-luteal period. Proof of ovarian cyclicity currently is mandatory before Species Survival Plan breeding recommendations are approved. However, because many adult females are not cycling normally, the reproductive monitoring of all cows throughout their life span is now encouraged. Complete endocrine evaluations in conjunction with ultrasound examinations and behavioral assessments are needed to identify causes of reproductive failure and develop mitigating treatments. Progestogen analyses also are effective for monitoring pregnancy, but only if longitudinal samples are collected. Alternatively, pregnancy can be diagnosed in occasional samples using serum prolactin or possibly relaxin measurements after 20 weeks of gestation. Parturition can be predicted on the basis of the rapid decrease in progestogens that occurs about 2–5 days before birth. An updated model of ovarian dynamics during the estrous cycle suggests that two waves of follicular development occur 3 weeks apart during the non-luteal phase, possibly under the control of follicle-stimulating hormone. Each follicular wave culminates in a luteinizing hormone (LH) surge, with the second surge inducing ovulation and corpus luteum formation. The functional significance of the first, anovulatory LH surge is under investigation, but from a practical perspective it can be used to schedule breeding (by artificial insemination or natural mating) to coincide with the ovulatory LH surge. Less is known about the reproductive biology of bulls, aside from the fact that musth is associated with dramatic changes in androgen secretion. Studies are needed to determine whether poor libido and inadequate semen quality observed in some mature elephants are due to testicular steroidogenic dysfunction. When blood samples cannot be collected for routine hormone analysis, gonadal activity can be monitored non-invasively through the measurement of excreted steroid metabolites (males: androgens; females: estrogens, progestogens) in urine and feces. Lastly, suggestions for future research priorities are provided. Zoo Biol 19:347–367, 2000.

Social factors influence ovarian acyclicity in captive African elephants (Loxodonta africana).

Nearly one-third of reproductive age African elephants in North America that are hormonally monitored fail to exhibit estrous cycle activity, which exacerbates the nonsustainability of the captive population. Three surveys were distributed to facilities housing female African elephants to determine how social and environmental variables contribute to cyclicity problems. Forty-six facilities returned all three surveys providing information on 90% of the SSP population and 106 elephants (64 cycling, 27 noncycling and 15 undetermined). Logistic analyses found that some physiological and social history variables were related to ovarian acyclicity. Females more likely to be acyclic had a larger body mass index and had resided longer at a facility with the same herdmates. Results suggest that controlling the weight of an elephant might be a first step to helping mitigate estrous cycle problems. Data further show that transferring females among facilities has no major impact on ovarian activity. Last, social status appears to impact cyclicity status; at 19 of 21 facilities that housed both cycling and noncycling elephants, the dominant female was acyclic. Further studies on how social and environmental dynamics affect hormone levels in free-living, cycling elephants are needed to determine whether acyclicity is strictly a captivity-related phenomenon.

Impact of social management on reproductive, adrenal and behavioural activity in the cheetah (Acinonyx jubatus)

Cheetahs (Acinonyx jubatus) held ex situ can provide an important resource for obtaining new biological information that usually cannot be gleaned from free-living individuals. However, consistent captive propagation of the cheetah, a prerequisite for establishing a self-sustaining population, has not been accomplished so far. This study examined the effect of a husbandry regimen commonly used in ex situ facilities on female cheetahs. Although generally solitary in the wild, zoos frequently house cheetahs in pairs or groups. Using non-invasive hormone monitoring and quantitative behavioural observations, we studied the impact of such enforced social conditions on behaviour and ovarian/adrenal activity. Eight female cheetahs were evaluated for two consecutive 6-month periods, first while maintained in pairs and then as individuals. Subsequently four females were regrouped into two new pairs and monitored for another 6 months. Females in five of six pairings demonstrated prolonged anoestrus and displayed agonistic behaviours. After pair separation all females rapidly resumed oestrous cyclicity. Females in the sixth pair continued cycling throughout the year while consistently displaying affiliative grooming and no agonistic behaviours. Faecal corticoid patterns varied significantly among individuals, but appeared unrelated to behavioural or ovarian hormone patterns. Thus, data appear to indicate that same-sex pair-maintenance of behaviourally incompatible female cheetahs may lead to suppressed ovarian cyclicity. This suppression appears linked to agonistic behaviours but not to any particular adrenal hormone excretion pattern. Results clearly demonstrate the value of applying knowledge about in situ social behaviour to ex situ management practices. Conversely, however, non-invasive hormone monitoring conducted ex situ may help us to identify physiological phenomena of potential relevance for future in situ studies.

Ultrasonography of the Urogenital Tract in Elephants (Loxodonta africana and Elephas maximus): An Important Tool for Assessing Male Reproductive Function

The success rate of captive elephant breeding programs worldwide is poor. Along with undiagnosed reproductive disorders in females and fatal diseases such as the newly discovered herpesvirus infection, male infertility now is considered a major contributing factor in the failure to maintain self-sustaining captive populations. To address questions related to male reproductive dysfunction, approximately 309 ultrasonographic assessments combined with semen collection were performed in captive (n = 10) and wild (n = 4) African (Loxodonta africana) and captive (n = 61) Asian (Elephas maximus) elephants. Bulls ranged from 4 to 50 years of age and were examined at 9 institutions in North America, 13 in Europe, 2 in Africa, and 7 in Asia. About half of the reproductive assessments were performed in protected contact situations with elephants handled in a restraint device, and half involved assessments of trained Asian bulls managed in free contact. Four wild African and two Asian elephant bulls were evaluated after receiving general anesthesia. Transrectal ultrasound was used to characterize the morphology and functionality of the entire urogenital tract, including the testes and accessory sex organs. Bulls were categorized on the basis of breeding status (breeders vs. non-breeders) and social history (i.e., type of interaction with conspecifics and keepers). Most of the bulls were non-breeders (designated Types I–V). Type I (n = 3 African, 6 Asian) and Type V (n = 1 Asian) were immature and castrate, respectively. On the basis of keeper evaluations, Type II bulls (n = 2, 4) were subordinate to older cows and keepers, whereas Type III bulls (n = 4, 28) were dominated by other bulls. Type IV (n = 1, 8) were older bulls of unknown history that exhibited numerous testicular pathologies resulting in poor semen quality. Non-breeding bulls included those that were exposed to females, but failed to breed, as well as those that had no opportunities to breed. Type VI individuals (n = 4, 14) were proven breeders. The percentage of observable reproductive tract pathology in adult males was remarkably low (14%), even in older bulls. However, apparent infertility of non-organic cause (i.e., not due to specific anatomical abnormalities) in these otherwise healthy bulls was high (32%). Semen quality varied markedly in ejaculates collected from the same bull, as well as from different bulls. In conclusion, although many of these bulls could serve as semen donors for natural mating or artificial insemination, the inconsistent production of good-quality ejaculates raises questions as to the reliability of these individuals to participate in breeding programs. The apparent inhibitory effect of suppressive social interactions on reproductive potential also needs to be investigated. Ultrasound examinations combined with semen collection should be conducted periodically to estimate the reproductive value of each bull and determine whether altered management strategies are needed to enhance captive breeding. Zoo Biol 19:333–345, 2000.

REPRODUCTIVE CHARACTERISTICS OF MALE FLORIDA PANTHERS: COMPARATIVE STUDIES FROM FLORIDA, TEXAS, COLORADO, LATIN AMERICA, AND NORTH AMERICAN ZOOS

Testicular volume, semen traits, and pituitary-gonadal hormones were measured in populations of Felis concolor from Florida, Texas, Colorado, Latin America, and North American zoos. More Florida panthers ( F. concolor coryi ) were unilaterally cryptorchid (one testicle not descended into the scrotum) than other populations (43.8 versus 3.9%, respectively). Florida panthers also had lower testicular and semen volumes, poorer sperm progressive motility, and more morphologically abnormal sperm, including a higher incidence of acrosomal defects and abnormal mitochondrial sheaths. Transmission electron microscopy revealed discontinuities in the acrosome, extraneous acrosomal material under the plasma membrane, and remnants of the golgi complex under the acrosome. No differences were detected in mean-circulating follicle-stimulating hormone, luteinizing hormone, or testosterone between Florida panthers and other populations of mountain lions. Seminal traits and concentrations of follicle-stimulating hormone, luteinizing hormone, and testosterone were similar between cryptorchid and noncryptorchid Florida panthers. Animals with F. concolor coryi ancestry were categorized on the basis of amount of genetic variation (low = type A; medium = type B; high = captive Piper stock). Compared to counterparts, type A Florida panthers had the lowest testicular volume and sperm-motility ratings and were the only animals exhibiting unilateral cryptorchidism. These results demonstrate the existence of major morphological and physiological differences among populations of F. concolor , a finding potentially related to differences in genetic diversity.

Chorionic gonadotropin administration in domestic cats causes an abnormal endocrine environment that disrupts oviductal embryo transport.

Fecal steroid analysis was used to investigate relationships between endocrine parameters and embryo characteristics in domestic cats subjected to chorionic gonadotropin stimulation and artificial insemination (AI). In Study 1, normal endocrine patterns were assessed in 12 cycling domestic queens. Fecal estradiol (E) patterns established an anovulatory cycle length of 18.3 +/- 0.4 d with estrus lasting 6.3 +/- 0.3 d. Eight females (67%) exhibited at least one spontaneous ovulation based on sustained increases in fecal progestagens (P). In Study 2, queens were mated during natural estrus (NE, n = 5) or subjected to exogenous i.m. gonadotropin stimulation, 100 IU eCG followed by 75 IU hCG 80 h later, (GS, n = 5). Compared with NE queens, fecal E concentrations were higher (P < 0.05) and remained elevated longer after ovulation induction with hCG. In Study 3, gonadotropin-stimulated queens (n = 7) were artificially inseminated and ovariohysterectomized 160 h after hCG. Ancillary follicles and/or corpora lutea were observed in 5 of 6 (83%) ovulating queens. Both fecal E and number of unovulated follicles observed at ovariohysterectomy were negatively correlated with the percentage of embryos recovered from the uterus (r = -0.91 and r = -0.87, respectively; P < 0.05). In summary, exogenous gonadotropin administration causes an abnormal endocrine environment in domestic cats, likely due to ancillary follicle development. The sustained elevations in estradiol appear to impair oviductal transport of embryos, possibly leading to the reduced fertility typically observed in cats subjected to gonadotropin stimulation and AI.

Effect of housing and environmental enrichment on adrenocortical activity, behavior and reproductive cyclicity in the female tigrina (Leopardus tigrinus) and margay (Leopardus wiedii)

The objective of this study was to evaluate the effects of different captive housing conditions on reproductive cyclicity and adrenocortical activity in adult females of two small-sized felid species, the tigrina (Leopardus tigrinus; n = 3) and margay (Leopardus wiedii; n = 2). Females were housed as singletons and subjected to three enclosure conditions over successive time periods: Phase I-large, enriched enclosures for 3 months; Phase II-small, empty enclosures for 5.5 months; Phase III-the same small enclosures enriched with branches and nest boxes for 6.5 months. Fecal samples were collected five times weekly throughout the study for analysis of progestagen, estrogen, and corticoid metabolites. On the basis of observed behaviors, stereotypic pacing was more frequent before feeding for all cats, regardless of enclosure conditions. Both species displayed a bimodal activity pattern, with peaks occurring at nightfall and dawn. All animals exhibited agitated behavior, characterized by a high frequency and duration of stereotypic pacing, primarily during the first 3 days after moving to the small empty enclosures. On the basis of hormonal analyses, ovarian follicular activity decreased and corticoid concentrations increased in tigrinas after transfer to the small barren cages compared to the patterns observed in the initial large, enriched enclosures. Corticoid concentrations in tigrinas then declined after small cage enrichment. Margay females exhibited increased corticoid excretion during Phases II and III, but in contrast to tigrinas, concentrations remained high even after cage enrichment. It was further showed that enriching the small enclosures was insufficient to reestablish normal ovarian activity within the time frame of the study for both species. In summary, margay and tigrina females exhibited distinct elevations in corticoid concentrations after transfer from large enriched enclosures to smaller barren cages that corresponded with agitated behavior, especially immediately after transfer. Fecal corticoid concentrations were reduced after cage enrichment in tigrinas, but not in margays. Although only a few individuals were evaluated, data suggest there may be species differences in response to captive environmental conditions. Overall results emphasize the importance of enclosure dimensions and enrichment when designing species appropriate environments for improving the health and reproductive fitness of threatened species. Zool Biol 26:441-460, 2007. (c) 2007 Wiley-Liss, Inc.