Establishing models of corticosteroid patterns during the life history of killer whales (Orcinus orca) under human care.

The use of corticosteroids and their metabolites as a physiologic measure of stress in wildlife species is increasing in both in and ex situ populations. However, factors such as season, circadian rhythm, sex and age are also known to influence corticosteroid production in wildlife. Our objective was to evaluate the influence of these variables on serum cortisol, corticosterone, aldosterone and their fecal metabolites in zoo-based killer whales (Orcinus orca). For evaluation of season, sex and age, we examined 30 animals (21 females, 9 males), and for circadian rhythm, we studied 18 animals (10 females, 8 males). Season did not influence corticosteroids (P ≥ 0.19). Circadian rhythm influenced all corticosteroids (P ≤ 0.012). Serum cortisol and corticosterone were highest in the morning and lower by mid-day and evening. Serum aldosterone was lowest during mid-day and highest in the evening. Excreted corticosteroid metabolites were higher in the morning and mid-day compared to evening (P ≤ 0.002). Serum cortisol was higher in males than females (P = 0.011). The inverse was observed for fecal corticosterone metabolites (P = 0.03). Serum corticosterone increased with age in all animals (P = 0.002), but serum cortisol increased with age in males only (P < 0.001). Finally, combining previously published data and these data reported herein, we developed models of corticosteroid patterns for zoo-based killer whales during life history events, including circadian variation, pregnancy and acute stress that may serve as a guide for evaluating stress physiology and animal welfare in this species.

Androgen and glucocorticoid production in the male killer whale (Orcinus orca): influence of age, maturity, and environmental factors.

Circulating concentrations of testosterone and its precursor androstenedione, as well as dehydroepiandrosterone (DHEA) and the adrenal hormones cortisol and corticosterone were measured at monthly intervals in 14 male killer whales (Orcinus orca) aged 0.8-38 years. Analyses were performed for examination of the relationships of age, sexual maturation status (STATUS), season, and environmental temperature (monthly air ambient temperature, A-TEMP) with hormone production using a mixed effects linear regression model with animal ID as the random variable. Hormone profiles, derived from enzyme immunoassay procedures validated herein, established that simultaneous up-regulation of androstenedione and testosterone production occurs at puberty, when males are aged 8-12 years. Androgen (testosterone and androstenedione) production in pubertal and adult males was influenced by season, with highest (p < 0.01) concentrations observed in spring and summer months. A significant effect of STATUS and season on DHEA production was also documented, with higher (p < 0.05) concentrations in pubertal and adult males compared to juvenile males, and higher (p < 0.05) concentrations in the months of summer than the fall. Among adult males (≥13 years), those classified as aged (≥31 years) had concentrations of testosterone and both glucocorticoids that were lower (p < 0.05), and those of androstenedione that were higher (p < 0.05) than their younger counterparts. The cortisol:corticosterone ratio for adult males was 7 : 1, and both glucocorticoids were affected by STATUS (p < 0.05), but not season or A-TEMP. Results of this research enhance our understanding of reproductive and adrenocortical function in healthy male killer whales and provide baseline profiles of hormone production for use in the species' health assessment and conservation.

Integrating neurocritical care approaches into neonatology: should all infants be treated equitably?

To improve the neurologic outcomes for infants with brain injury, neonatal providers are increasingly implementing neurocritical care approaches into clinical practice. Term infants with brain injury have been principal beneficiaries of neurologically-integrated care models to date, as evidenced by the widespread adoption of therapeutic hypothermia protocols for hypoxic-ischemic encephalopathy. Innovative therapeutic and diagnostic support for very low birth weight infants with brain injury has lagged behind. Given that concern for significant future neurodevelopmental impairment can lead to decisions to withdraw life supportive care at any gestational age, providing families with accurate prognostic information is essential for all infants. Current variable application of multidisciplinary neurocritical care approaches to infants at different gestational ages may be ethically problematic and reflect distinct perceptions of brain injury for infants born extremely premature.

Sperm sex-sorting and preservation for managing the sex ratio and genetic diversity of the southern white rhinoceros (Ceratotherium simum simum).

White rhinoceros ejaculates (n=9) collected by electroejaculation from four males were shipped (10°C, 12h) to develop procedures for the production of chilled and frozen-thawed sex-sorted spermatozoa of adequate quality for artificial insemination (AI). Of all electroejaculate fractions, 39.7% (31/78) exhibited high quality post-collection (≥70% total motility and membrane integrity) and of those, 54.8% (17/31) presented reduced in vitro quality after transport and were retrospectively determined to exhibit urine-contamination (≥21.0µg creatinine/ml). Of fractions analyzed for creatinine concentration, 69% (44/64) were classified as urine-contaminated. For high quality non-contaminated fractions, in vitro parameters (motility, velocity, membrane, acrosome and DNA integrity) of chilled non-sorted and sorted spermatozoa were well-maintained at 5°C up to 54h post-collection, whereby >70% of post-transport (non-sorted) or post-sort (sorted) values were retained. By 54h post-collection, some motility parameters were higher (P<0.05) for non-sorted spermatozoa (total motility, rapid velocity, average path velocity) whereas all remaining motion parameters as well as membrane, acrosome and DNA integrity were similar between sperm types. In comparison with a straw method, directional freezing resulted in enhanced (P<0.05) motility and velocity of non-sorted and sorted spermatozoa, with comparable overall post-thaw quality between sperm types. High purity enrichment of X-bearing (89±6%) or Y-bearing (86±3%) spermatozoa was achieved using moderate sorting rates (2540±498X-spermatozoa/s; 1800±557Y-spermatozoa/s). Collective in vitro characteristics of sorted-chilled or sorted-frozen-thawed spermatozoa derived from high quality electroejaculates indicate acceptable fertility potential for use in AI.

Development and evaluation of deep intra-uterine artificial insemination using cryopreserved sexed spermatozoa in bottlenose dolphins (Tursiops truncatus).

Since its development in bottlenose dolphins, widespread application of AI with sex-selected, frozen-thawed (FT) spermatozoa has been limited by the significant expense of the sorting process. Reducing the total number of progressively motile sperm (PMS) required for an AI would reduce the sorting cost. As such, this research compared the efficacy of small-dose deep uterine AI with sexed FT spermatozoa (SEXED-SMALL; ~50×10(6)PMS, n=20), to a moderate dose deposited mid-horn (SEXED-STD, ~200×10(6)PMS; n=20), and a large dose of FT non-sexed spermatozoa deposited in the uterine body (NONSEXED-LARGE, 660×10(6)PMS, n=9). Ten of the 11 calves resulting from use of sexed spermatozoa were of the predetermined sex. Similar rates of conception (NONSEXED-LARGE: 78%, SEXED-STD: 60%, SEXED-SMALL: 57%) and total pregnancy loss (TPL: NONSEXED-LARGE: 28.6%; SEXED-STD: 41.0%; SEXED-SMALL: 63.6%) were observed across groups, but early pregnancy loss (EPL,

Sperm DNA fragmentation and morphological degeneration in chilled elephant (Elephas maximus and Loxodonta Africana) semen collected by transrectal massage.

Ejaculates from nine Asian and two African elephants were analysed to gain a further understanding of mechanisms underlying variable semen quality after transrectal massage. Semen analysis was performed after collection (0 h; subjective motility parameters only) and after 24 h of chilled storage at 10 °C (24 h; all ejaculate and sperm characteristics). Ejaculates with ≤50% total motility (TM) at 24 h, which represented >90% of collection attempts, contained a sperm population with a high degree of DNA damage (64.2 ± 19.2% fragmented DNA) and an elevated incidence of detached heads (43.3 ± 22.5%). In contrast, good quality ejaculates designated as those with >50% TM at 24 h displayed higher (p < 0.05) values of sperm kinetic parameters, DNA integrity and normal morphology. Fertility potential was high for good quality ejaculates from two males (one Asian and one African bull) based on in vitro characteristics after chilled storage for up to 48 h post-collection. Urine contamination of semen, as assessed quantitatively by creatinine concentration, was confirmed as a significant factor in reduced elephant ejaculate quality. However, the identification of considerable DNA damage and morphological degeneration in the majority of ejaculates after only 24 h of chilled storage indicates that sperm ageing could be a primary contributor to inconsistent semen quality in the elephant.

Characteristics of high-quality Asian elephant (Elephas maximus) ejaculates and in vitro sperm quality after prolonged chilled storage and directional freezing.

The in vitro quality of spermatozoa from one elephant (Elephas maximus) was examined after chilled storage and directional freezing (DF). High-quality, non-contaminated ejaculates (77.6±6.0% progressive motility, 3.9±1.5 µg creatinine mL(-1) raw semen, 2.7±0.6% detached heads) were cryopreserved after 0 (0hStor), 12 (12hStor) and 24 h (24hStor) of chilled storage. At 0 h and 6h post-thawing, total motility, plasma membrane integrity, acrosome integrity, mitochondrial activity and normal morphology were similar (P>0.05) across treatments. In contrast, progressive motility, rapid velocity and several kinematic parameters were lower (P<0.05) for 24Stor compared with 0hStor at 0 h post-thaw. By 6 h post-thaw, amplitude of lateral head displacement and velocity parameters (average pathway, straight-line and curvilinear velocity) were lower (P<0.05) for 24hStor compared with 0hStor and 12hStor. DNA integrity was high and remained unchanged (P>0.05) across all groups and processing stages (1.6±0.6% of cells contained fragmented DNA). Results indicate that DF after up to 12 h of chilled storage results in a post-thaw sperm population of acceptable quality for artificial insemination. These findings have implications for the cryopreservation of sex-sorted spermatozoa, which typically undergo more than 12 h of chilled storage prior to sorting and preservation.

Characterization of the estrous cycle in female beluga (Delphinapterus leucas) using urinary endocrine monitoring and transabdominal ultrasound: Evidence of facultative induced ovulation.

Recent, successful application of assisted reproductive technologies in captive beluga has resulted from the extensive study of male beluga reproductive biology. Optimization of assisted reproduction requires additional detailed knowledge of the female estrous cycle. Our specific objectives were to: (1) validate urinary immunoassays for use in this species; (2) elucidate annual ovarian cycle dynamics through the combined use of hormone excretion patterns and transabdominal ultrasound; and (3) establish whether ovulation in this species is spontaneous or induced by male factors. Ovulation was observed in four of 15 estrous cycles monitored in four adult female beluga maintained in a single-sex group. After introduction of a breeding male, ovulation was observed in six of seven estrous cycles. All estrous cycles occurred from March through June. For spontaneous ovulations (n=4), the inter-estrous interval was 34d (range 33-35d), with a follicular phase length (FPL) of 25±8d (mean±SD). For all ovulatory estrous cycles (with and without a breeding male), urinary estrogen conjugates (EC, 15.3±7.9ng/mg Cr) and ovulatory luteinizing hormone (ovLH, 17.1±6.6ng/mg Cr) concentrations both peaked on Day 0, and EC concentrations returned to baseline 8±7d later. For non-conceptive cycles, urinary progestagen (Pg) concentrations increased on Day 0 (3.5±1.7ng/mg Cr), peaked on Day+19 (19.7±17.1ng/mg Cr), and were elevated above baseline for 27±4d. Preovulatory follicular diameter and circumference on Day -2±2 (range: Day -4 to -1) from peak EC were 2.5±0.7 and 7.8±1.3cm, respectively. The FPL in non-ovulatory estrous cycles (n=11) lasted 24±10d and EC concentrations gradually declined to baseline over a 21±10d interval following the EC peak (27.8±28.8ng/mg Cr). Non-ovulatory estrous cycles were characterized by the absence of an ovLH surge and no concomitant increase in Pg concentrations above baseline excretion; the mean follicular diameter at or near peak EC was 3.1±0.8cm on Day 2 ±3d from peak EC (range: -1 to +5days from peak EC). Overall, these data confirm that captive beluga exhibit reproductive seasonality and demonstrate that the species is a facultative-induced ovulator.

In vitro sperm characterization and development of a sperm cryopreservation method using directional solidification in the killer whale (Orcinus orca).

Research was conducted to characterize seminal traits and to develop a sperm cryopreservation method using directional freezing (DF) for the killer whale (Orcinus orca). Experiments evaluated effects of: (i) freezing rate (SLOW, MED, FAST) by diluent (BF5F, Biladyl®, EYC) in 0.5 mL straws; and (ii) freezing method (straw or DF) by glycerol (3, 6, or 9% final concentration, v:v) on in vitro sperm quality. Fresh ejaculates (n = 161) were (mean ± SD) 7.8 ± 7.4 mL at 740 × 10(6) sperm/mL with 92.2 ± 6.3% total motility (TM), 85.4 ± 6.9% progressive motility (PM), 89.6 ± 9.0% viability and 89.8 ± 9.2% acrosome integrity. Samples frozen using straws by the MED or SLOW method were improved (P < 0.05) over FAST across all diluents. At 3 h post thaw (PT), TM, PM, Rapid motility (RM), VAP, VCL, ALH and viability for 3% and 6% glycerol were improved (P < 0.05) over 9% glycerol. Directional freezing samples at 0 h and 3 h PT, at all glycerol concentrations, displayed higher (P < 0.001) TM, PM, RM, VAP, VSL, VCL and viability /intact acrosomes (PI/FITC-PNA) than straw. These data provided the first information on ejaculate characteristics and the development of a semen cryopreservation method using DF in the killer whale.

Deep intra-uterine artificial inseminations using cryopreserved spermatozoa in beluga (Delphinapterus leucas).

Artificial insemination (AI) with liquid-stored spermatozoa and sperm cryopreservation using directional freezing (DF) have been successful in the beluga. This study built on this foundation to develop a deep intra-uterine AI technique with frozen-thawed semen in beluga. Forty-two ejaculates from one male were cryopreserved using DF technology and subsequently used for 10 insemination attempts with seven females. Percentage pre- and post-thaw progressive motility and viability were (mean +/- SD) 73.0 +/- 12.2, 38.4 +/- 8.8, 88.0 +/- 0.1, and 59.3 +/- 15.7%, respectively. A series of GnRH injections (3 x 250 microg, IV, 1.5 to 2 h apart) were used to induce ovulation, once a growing follicle >2.5 cm in diameter was visualized via trans-abdominal ultrasonography. Artificial insemination was performed at 30.1 +/- 3.8 h post-initial GnRH injection with semen deposited in the uterine horn, 92.6 +/- 16.2 cm beyond the genital opening using a flexible endoscope. The external cervical os (cEOS) was located beyond a series of 5 to 10 vaginal rings, 44.8 +/- 9.3 cm from the external genital opening. The internal bifurcation of the uterus was 27 +/- 6.8 cm beyond the cEOS. Ovulation occurred at 8.5 +/- 7.6 h post-AI. Two of 10 inseminations (20%) resulted in pregnancy. The first pregnancy resulted in twins; both calves were born 442 d after AI, with one surviving. The second pregnancy is ongoing. These findings represent the first successful application of AI using frozen-thawed semen in beluga, and are important examples of how assisted reproductive technologies can provide tools for the global management of threatened species.

Seasonality, estrous cycle characterization, estrus synchronization, semen cryopreservation, and artificial insemination in the Pacific white-sided dolphin (Lagenorhynchus obliquidens).

The reproductive physiology of the Pacific white-sided dolphin, Lagenorhynchus obliquidens, was characterized to facilitate the development of artificial insemination (AI) using cryopreserved spermatozoa. Specific objectives were to: 1) describe reproductive seasonality of the Pacific white sided dolphins; 2) describe urinary LH and ovarian steroid metabolites during the estrous cycle; 3) correlate LH and ovarian steroidal metabolite patterns to ultrasound-monitored follicular growth and ovulation; and 4) assess the efficacy of synchronizing estrus, sperm collection/cryopreservation, and intrauterine insemination. Ovulations (64%, n=37) and conceptions (83%, n=18) occurred from August to October. Peak mean serum testosterone (24 ng/ml), cross-sectional testicular area (41.6 cm(2)), and sperm concentration (144.3 x 10(7) sperm/ml) occurred in July, August, and September respectively. Spermatozoa were only found in ejaculates from July to October. Estrous cycles (n=22) were 31 d long and were comprised of a 10 d follicular and 21 d luteal phase. Ovulation occurred 31.2 h after the onset of the LH surge and 19.3 h after the LH peak. Follicular diameter and circumference within 12 h of ovulation were 1.52 and 4.66 cm respectively. Estrus synchronization attempts with altrenogest resulted in 17 (22%) ovulatory cycles with ovulation occurring 21 d post-altrenogest. Ten AI attempts using cryopreserved semen resulted in five pregnancies (50%). The mean gestation length was 356 days (range 348-367). These data provide new information on the Pacific white-sided dolphin's reproductive physiology and collectively enabled the first application of AI in this species.

Application of sperm sorting and associated reproductive technology for wildlife management and conservation.

Efforts toward the conservation and captive breeding of wildlife can be enhanced by sperm sorting and associated reproductive technologies such as sperm cryopreservation and artificial insemination (AI). Sex ratio management is of particular significance to species which naturally exist in female-dominated social groups. A bias of the sex ratio towards females of these species will greatly assist in maintaining socially cohesive groups and minimizing male-male aggression. Another application of this technology potentially exists for endangered species, as the preferential production of females can enable propagation of those species at a faster rate. The particular assisted reproductive technology (ART) used in conjunction with sperm sorting for the production of offspring is largely determined by the quality and quantity of spermatozoa following sorting and preservation processes. Regardless of the ART selected, breeding decisions involving sex-sorted spermatozoa should be made in conjunction with appropriate genetic management. Zoological-based research on reproductive physiology and assisted reproduction, including sperm sorting, is being conducted on numerous terrestrial and marine mammals. The wildlife species for which the technology has undergone the most advance is the bottlenose dolphin. AI using sex-sorted fresh or frozen-thawed spermatozoa has become a valuable tool for the genetic and reproductive management of captive bottlenose dolphins with six pre-sexed calves, all of the predetermined sex born to date.

Semen collection, characterisation and artificial insemination in the beluga (Delphinapterus leucas) using liquid-stored spermatozoa.

Ejaculates were collected from a beluga (Delphinapterus leucas) to gain an understanding of sperm biology and develop a short-term sperm preservation method for use in artificial insemination (AI). Ejaculate parameters and biochemistry, semen production and serum testosterone concentrations of an adult male were characterised for 21 months. Sperm viability, acrosome integrity and morphology did not change (P > 0.05) but ejaculate volume, sperm concentration and total spermatozoa per ejaculate were higher (P < 0.05) from January to June than from July to December. Peak testosterone concentrations (P < 0.05) were observed from October to April (8.0 +/- 1.6 ng mL(-1)). The effects of hyaluronic acid (HA), antioxidants, storage temperature and time on in vitro sperm characteristics were examined. Motility parameters and viability were improved (P < 0.05) when semen was stored at 5 degrees C compared with 21 degrees C. During the first 24 h of storage sperm agglutination was absent only at 5 degrees C in the presence of HA. A nulliparous 28-year-old female was inseminated endoscopically with liquid-stored semen. A pregnancy and birth of a calf was achieved following AI for the first time in this species, thereby validating both the AI technique and the fertility of beluga spermatozoa after chilled storage in a specialised diluent.

Estrous cycle characterisation and artificial insemination using frozen-thawed spermatozoa in the bottlenose dolphin (Tursiops truncatus).

The reproductive endocrinology of the bottlenose dolphin, Tursiops truncatus, was characterized to facilitate the development of artificial insemination using cryopreserved spermatozoa. Specific objectives were: (i) to determine the excretory dynamics of urinary luteinizing hormone (LH) and ovarian steroid metabolites during the estrous cycle; (ii) to evaluate the effect of an exogenously administered synthetic progesterone analog (altrenogest) on reproductive hormone excretion; (iii) to correlate follicular growth and ovulation (as determined by transabdominal ultrasound) to urinary LH and ovarian steroid metabolites; (iv) examine the in vivo fertilisation capacity of cryopreserved semen, and (v) to develop an intrauterine insemination technique. Based on urinary endocrine monitoring of natural estrous cycles (2 consecutive cycles) and nine post altrenogest cycles in ten females, estrous cycles were found to be 36 days long and comprised of an 8 day and 19 day follicular and luteal phase, respectively. Peak estrogen conjugates (EC; 5.4+/-3.8 ng/mg creatinine (Cr)) occurred 8 h prior to the LH surge (70.9+/-115.7 ng/mg Cr). The time of ovulation, as determined by ultrasonography, occurred 32.1+/-8.9 h and 24.3+/-7.0 h after the onset of the LH surge and LH peak, respectively. Mean preovulatory follicular diameter and circumference were 2.1+/-0.5 cm and 6.5+/-1.5 cm, respectively. Of the 27 estrous synchronisation attempts, 13 resulted in an ovulatory cycle, with ovulation occurring 21 days post-altrenogest treatment. Intrauterine (4 of 5) and intracornual (1 of 3) inseminations conducted across eight estrous cycles resulted in five pregnancies (63%), one pregnancy resulted from the use of liquid stored semen, whereas four were achieved using cryopreserved semen. These data provide new information on female bottlenose dolphin reproductive physiology, and demonstrate that the combination of endocrine monitoring and serial ultrasonography contributed to successful AI using liquid-stored and cryopreserved semen.

Reproductive physiology and development of artificial insemination technology in killer whales (Orcinus orca).

Research was conducted to define the basic reproductive physiology of killer whales (Orcinus orca) and to use this knowledge to facilitate the development of artificial insemination procedures. The specific objectives were 1) to determine the excretory dynamics of urinary LH and ovarian steroid metabolites during the estrous cycle; 2) to evaluate the effect of an exogenously administered, synthetic progesterone analog on reproductive hormone excretion; 3) to validate the use of transabdominal ultrasound for ovarian evaluation and timing of ovulation; 4) to examine the quality of semen after liquid storage and cryopreservation; and 5) to develop an intrauterine insemination technique. Based on urinary endocrine monitoring of 41 follicular phases and 26 complete cycles from five females, estrous cycles were 41 days long and comprised a 17-day follicular phase and a 21-day luteal phase. A consistent temporal relationship was observed between peak estrogen conjugates and the LH surge, the latter of which occurred approximately 0.5 days later. Two animals placed on oral altrenogest (three separate occasions for 30, 17, and 31 days, respectively) excreted peak urinary estrogen concentrations 25 days after withdrawal that were followed by sustained elevations in urinary pregnanediol-3alpha-glucuronide excretion. Mean preovulatory follicle diameter was 3.9 cm (n = 6), and ovulation occurred 38 h (n = 5) after the peak of the LH surge. Based on visual estimates of motility, liquid-stored semen maintained 92% of its raw ejaculate sperm motility index (total progressive motility x kinetic rating [0-5 scale, where 0 = no movement and 5 = rapid progressive movement]) when held at 4 degrees C for 3 days postcollection. Semen cryopreserved using a medium freezing rate demonstrated good postthaw total motility (50%), progressive motility (94%), and kinetic rating (3.5). Insemination during eight estrous cycles resulted in three pregnancies (38%), two from liquid-stored and one from cryopreserved semen. Two calves were delivered after gestation lengths of 552 and 554 days, respectively. These data demonstrate the potential of noninvasive endocrine monitoring combined with serial ultrasonography to improve our understanding of the reproductive biology of cetaceans. This fundamental knowledge was essential for ensuring the first successful conceptions, resulting in live offspring, using artificial insemination in any cetacean species.

Disentangling measures of individual perceptions of community social dynamics: results of a community survey.

This study examined how different measures of individual perceptions of community social dynamics relate to each other and how these measures relate to self-reported general health and depressive symptoms. Results of a principal components analysis conducted to investigate the interrelationships between these individual measures suggest that these measures measure separate phenomena. In addition, in results of multiple-regression analyses conducted to examine associations between the various measures of individual perceptions of community social dynamics and the dependent variables of self-reported general health and depressive symptoms, sense of community, perceived neighborhood control, and neighborhood participation were all associated with the outcome variables in separate regression models. In a regression model with these three variables added to control variables, only sense of community was significantly, albeit modestly, associated with depressive symptoms and self-reported general health.