Factors Associated with Reproductive Success in Captive Vancouver Island Marmots (Marmota vancouverensis).

The Vancouver Island marmot (Marmota vancouverensis) is Canada's most endangered endemic mammal. In 1997, a conservation breeding-for-release program was established to supplement wild marmot populations. Retrospective analyses of captive breeding studbook records since 2000 indicate the age of the sire and the dam significantly impacted the odds of successfully weaning a litter. Dams and sires between 5 and 7 years of age had more than double the odds of reproductive success compared to older animals. Successful reproduction by the dam in the previous year also doubled the odds of successfully weaning a litter in subsequent years. Assessment of adrenal function via fecal glucocorticoid analyses indicated established breeding pairs had decreased stress compared to new pairs (5.74 ± 0.28 ng/g vs. 7.60 ± 0.34 ng/g; p < 0.0001). Pairs that were ultimately successful at weaning pups in a breeding season had decreased stress compared to unsuccessful pairs (6.05 ± 0.34 ng/g vs. 7.22 ± 0.28 ng/g; p = 0.0006). These endocrine results suggest social buffering via familiarity and breeding/pair bond formation may be decreasing stress in established and successful pairs, respectively. The results of this study will be used to assist in the captive breeding management of this species to optimise numbers of animals produced to supplement the wild populations.

The Adrenal Cortisol Response to Increasing Ambient Temperature in Polar Bears (Ursus maritimus).

Our objective was to identify the upper ambient temperature threshold that triggers an increase in cortisol in response to increased thermoregulatory demands in polar bears. The results reported here include endocrine data collected over two years from 25 polar bears housed in 11 accredited zoological institutions across North America. The effects of ambient temperature, sex, age group (juvenile, adult, elderly), breeding season and humidity on fecal cortisol metabolite (FCM) concentrations (N = 8439 samples) were evaluated using linear mixed models. Ambient temperatures were placed into five different categories: <5 °C, 6−10 °C, 11−15 °C, 16−20 °C, and >20 °C. Ambient temperature and humidity had a significant (p < 0.05) effect on FCM concentrations with significant (p < 0.05) interactions of sex, age and breeding season. Once biotic factors were accounted for, there was a significant (p < 0.05) increase in FCM concentrations associated with ambient temperatures above 20 °C in adult polar bears. The implications of these findings for the management of both zoo and wild polar bears are discussed.

A simple, field-friendly technique for cryopreserving semen from Asian elephants (Elephas maximus).

The specific objectives of the present study were to investigate the effects of manual seeding, differing freeze and thaw rates as well as storage for 24h at 4°C prior to cryopreservation on post-thaw sperm quality in Asian elephants. Extended semen was cooled in an equitainer to 4°C, frozen in liquid nitrogen vapour at various rates with and without manual seeding or in a dry shipper and thawed at 37, 50 and 75°C. There was a significant effect of freeze rate on post-thaw motility (P<0.0001) and acrosomal integrity (P<0.005). The faster freeze rates in the dry shipper and at 1cm or 2cm above liquid nitrogen consistently provided better cryopreservation than slower freezing rates. Thaw temperature had no effect on post-thaw semen quality but there was an interaction between freeze and thaw rates with higher thaw rates resulting in superior post-thaw semen quality in straws frozen at fast rates. Storage of samples prior to freezing had a detrimental effect on post-thaw semen quality. In summary, our results indicate cooling extended semen in an equitainer and cryopreserving it by placing straws directly in a dry shipper is a simple technique for effectively cryopreserving Asian elephant semen in the field or zoo.

Seasonal variation in urinary and salivary reproductive hormone levels in Amazonian manatees (Trichechus inunguis).

The Amazonian manatee (Trichechus inunguis) is a threatened aquatic mammal endemic to the Amazon basin. The aim of this study was to evaluate the urinary and salivary reproductive hormone levels of captive Amazonian manatees collected during two seasons of the year. Salivary samples from four males and urinary and salivary samples from three females were collected during two seasons (March-June and September-November) over two consecutive years. Salivary testosterone in males was measured by radioimmunoassay and reproductive hormones in females (salivary progesterone and oestradiol and urinary progestogens, oestrogens and luteinising hormone) were measured by enzyme immunoassay. The data were analysed in a 2×2 factorial design, where the factors were year and season. There was no effect of year or season for salivary testosterone. All female hormones showed a seasonal effect (higher hormone levels during March-June than September-November) or an interaction between year and season (P<0.05). These results strongly indicate the existence of reproductive seasonality in Amazonian manatees; however, apparently only females exhibit reproductive quiescence during the non-breeding season. Further long-term studies are necessary to elucidate which environmental parameters are related to reproductive seasonality in T. inunguis and how this species responds physiologically to those stimuli.

First attempt to monitor luteinizing hormone and reproductive steroids in urine samples of the Amazonian manatee (Trichechus inunguis).

The aims of this study were to validate an enzyme immunoassay (EIA) for the measurement of luteinizing hormone (LH) in urine samples of Amazonian manatees (Trichechus inunguis; Mammalia: Sirenia) and to monitor urinary LH and reproductive steroids during the ovarian cycle in this species. Urine samples were collected from two captive males following a hormonal challenge with a gonadotropin-releasing hormone (GnRH) analogue. The urinary LH results from hormonal challenge were compared with urinary androgens for the purpose of EIA validation. Furthermore, urine samples were collected daily, over a 12-wk period, from two captive adult females, for 2 consecutive yr. The urinary LH pattern from females was compared with the patterns of urinary progestagens and estrogen conjugates throughout the ovarian cycle. An LH peak was observed in both male Amazonian manatees after the hormonal challenge, occurring prior to or together with peak androgen levels. In the females, the ovarian cycle ranged from 40 to 48 days (mean of 43.7 days). Two distinct peaks of estrogen conjugates were observed across all cycles analyzed, and the urinary LH peaks observed were accompanied by peaks of urinary estrogen conjugates. The EIA was validated as a method for the quantification of urinary LH from Amazonian manatees, as it was able to detect variations in the levels of LH in urine samples. These results suggest that T. inunguis exhibits a peculiar hormonal pattern during the ovarian cycle. Therefore, further studies are desirable and necessary to clarify the relationship between this hormonal pattern and morphological changes, as well as mating behavior, in Amazonian manatee.

Efficacy of porcine gonadotropins for repeated stimulation of ovarian activity for oocyte retrieval and in vitro embryo production and cryopreservation in Siberian tigers (Panthera tigris altaica).

A comparison of the amino acid sequences demonstrated that Siberian tiger gonadotropins are more homologous with those of porcine than any other commercially available preparation. The present study measured the efficacy of repeated ovarian stimulation with purified porcine gonadotropins on the follicular, hormonal, and immunogenic responses in Siberian tigers as well as on the ability of oocytes retrieved by laparoscopic follicular aspiration to fertilize and cleave in vitro. Controlled rate and vitrification cryopreservation methods were also compared for their ability to support ongoing cleavage following thawing of presumptive 2- to 4-cell tiger embryos generated in vitro. Vitrification supported continued embryonic cleavage in vitro while controlled rate freezing did not. Stereological microscopy indicated an excellent ovarian response with the recovery of quality cumulus-oocyte complexes that apparently fertilized and cleaved in vitro. However, ultrastructural and physiological examination revealed abnormal and unnatural responses such as the failure of some cumulus-oocyte complexes to reach maturity and progestagen levels to approach normalcy. At the same time, analyses of blood for antibodies failed to detect an immune reaction to these foreign gonadotropins in an assay that tested positive for the chorionic gonadotropin-stimulated domestic cat. Together, these observations suggest that porcine gonadotropins may be effective for the ovarian stimulation of tigers but that some modifications to administration protocols are needed to produce a more natural response.

Endocrine patterns associated with reproduction in the Nile hippopotamus (Hippopotamus amphibius) as assessed by fecal progestagen analysis.

The hippopotamus (Hippopotamus amphibius) is a popular zoo animal and an integral part of wetland ecosystems in its native continent of Africa. However, information about the reproductive physiology of the hippopotamus is limited compared to the other megaherbivores of Africa such as the elephant and the rhinoceros. This study is the first to report on the endocrine patterns associated with ovulation and pregnancy in the Nile hippopotamus. Fecal samples were collected 3 times per week from female hippopotami (N = 9) housed in captivity in Florida. Progesterone metabolites were extracted from the feces with 80% MeOH and quantified using a progestagen EIA. Fecal progestagen analysis indicated hippopotami had a cycle length of 35.3 +/- 1.3 days and ovulated throughout the year. Fecal progestagen concentrations during pregnancy (N = 11 pregnancies) were higher on average (833.5 +/- 115.2 ng/g; P < 0.05) than those observed during non-pregnant luteal phases (309.5 +/- 20.9 ng/g) and remained elevated throughout gestation. Fecal progestagen analysis revealed the onset of puberty in three females between 3 and 4 years of age and all conceived by their fourth cycle. Lactational suppression of ovulation during nursing was usually, but not always, observed and lasted for 33.9 +/- 1.8 weeks. The observed calving interval in females with lactational anovulation was 17 months. The majority of births (63.6%) occurring during the summer rainy season and the inconsistency of lactational anovulation suggested the possibility of environmental influences on reproduction.