Anti-Müllerian hormone in managed African and Asian rhino species.

Serum collected across the lifespan of four managed rhino species: black (Diceros bicornis, n = 16), white (Ceratotherium simum simum, n = 19), greater one-horned (GOH, Rhinoceros unicornis, n = 11) and Sumatran (Dicerorhinus sumatrensis, n = 6) were validated and analyzed in an anti-Müllerian hormone (AMH) enzyme- linked immunoassay. Concentrations of AMH were examined over time, between sexes and throughout different reproductive states which included n = 3 female white rhinos immunocontracepted with porcine zona pellucida (pZP). Across species, males produced higher AMH concentrations compared to females. Among males, AMH concentrations varied by species aside from comparable values secreted between black and white rhinos. The GOH and Sumatran rhino secreted the highest and lowest male AMH concentrations, respectively. However, within each species, AMH concentrations were similar across male age categories. Preliminary insight into male AMH changes from birth to sexual maturity suggest its potential as a marker for onset of testicular maturation. Female black, GOH and Sumatran rhinos secreted comparable AMH concentrations which were higher than those in white rhino. Within each species, inter-individual variation in AMH secretion occurred among females of similar age. While AMH secretion did not differ across the ages sampled for female white (4->26 yr) and GOH (4-26 yr) rhinos, black and Sumatran rhinos >26 and <4 yr, respectively secreted lower AMH compared to conspecific females 7-26 yr of age. Two idiopathic infertility cases corresponded to low (outside species range) AMH values. The establishment of normative AMH concentrations in managed African and Asian rhinos provides an additional metric beyond traditional sex steroids to assess gonadal function. Further work is needed to determine if AMH can predict fertility potential in rhinos.

Epigenetic mechanisms in alcohol- and adversity-induced developmental origins of neurobehavioral functioning.

The long-term effects of developmental alcohol and stress exposure are well documented in both humans and non-human animal models. Damage to the brain and attendant life-long impairments in cognition and increased risk for psychiatric disorders are debilitating consequences of developmental exposure to alcohol and/or psychological stress. Here we discuss evidence for a role of epigenetic mechanisms in mediating these consequences. While we highlight some of the common ways in which stress or alcohol impact the epigenome, we point out that little is understood of the epigenome's response to experiencing both stress and alcohol exposure, though stress is a contributing factor as to why women drink during pregnancy. Advancing our understanding of this relationship is of critical concern not just for the health and well-being of individuals directly exposed to these teratogens, but for generations to come.

Sex Differences in Early Postnatal Microglial Colonization of the Developing Rat Hippocampus Following a Single-Day Alcohol Exposure.

Microglia are involved in various homeostatic processes in the brain, including phagocytosis, apoptosis, and synaptic pruning. Sex differences in microglia colonization of the developing brain have been reported, but have not been established following alcohol insult. Developmental alcohol exposure represents a neuroimmune challenge that may contribute to cognitive dysfunction prevalent in humans with Fetal Alcohol Spectrum Disorders (FASD) and in rodent models of FASD. Most studies have investigated neuroimmune activation following adult alcohol exposure or following multiple exposures. The current study uses a single day binge alcohol exposure model (postnatal day [PD] 4) to examine sex differences in the neuroimmune response in the developing rat hippocampus on PD5 and 8. The neuroimmune response was evaluated through measurement of microglial number and cytokine gene expression at both time points. Male pups had higher microglial number compared to females in many hippocampal subregions on PD5, but this difference disappeared by PD8, unless exposed to alcohol. Expression of pro-inflammatory marker CD11b was higher on PD5 in alcohol-exposed (AE) females compared to AE males. After alcohol exposure, C-C motif chemokine ligand 4 (CCL4) was significantly increased in female AE pups on PD5 and PD8. Tumor necrosis factor-α (TNF-α) levels were also upregulated by AE in males on PD8. The results demonstrate a clear difference between the male and female neuroimmune response to an AE challenge, which also occurs in a time-dependent manner. These findings are significant as they add to our knowledge of specific sex-dependent effects of alcohol exposure on microglia within the developing brain.

Monitoring and controlling ovarian function in the rhinoceros.

Despite their size and potentially dangerous demeanor, the rhinoceros has been a preferred subject of wildlife reproductive scientists. Several factors contribute to this taxon's popularity including the ability to utilize insightful tools like non-invasive hormone metabolite monitoring and transrectal ultrasonography, the necessity for mate introductions to coincide with the female's estrus when breeding certain species or individuals, and the desire to develop assisted reproductive technologies to facilitate the genetic management and ultimate sustainability of small, managed populations in human care. The resulting profusion of rhinoceros reproductive studies has revealed significant species-specific characteristics and exposed the prevalence of aberrant reproductive activity within this taxon. Of equal importance, it has guided necessary intervention and enhanced our success in overcoming challenges associated with breeding rhinoceroses.

Impact of exercise and a complex environment on hippocampal dendritic morphology, Bdnf gene expression, and DNA methylation in male rat pups neonatally exposed to alcohol.

Alcohol exposure in utero can result in Fetal Alcohol Spectrums Disorders (FASD). Measures of hippocampal neuroplasticity, including long-term potentiation, synaptic and dendritic organization, and adult neurogenesis, are consistently disrupted in rodent models of FASD. The current study investigated whether third trimester-equivalent binge-like alcohol exposure (AE) [postnatal days (PD) 4-9] affects dendritic morphology of immature dentate gyrus granule cells, and brain-derived neurotrophic factor (Bdnf) gene expression and DNA methylation in hippocampal tissue in adult male rats. To understand immediate impact of alcohol, DNA methylation was measured in the PD10 hippocampus. In addition, two behavioral interventions, wheel running (WR) and environmental complexity (EC), were utilized as rehabilitative therapies for alcohol-induced deficits. AE significantly decreased dendritic complexity of the immature neurons, demonstrating the long-lasting impact of neonatal alcohol exposure on dendritic morphology of immature neurons in the hippocampus. Both housing conditions robustly enhanced dendritic complexity in the AE animals. While Bdnf exon I DNA methylation was lower in the AE and sham-intubated animals compared with suckle controls on PD10, alterations to Bdnf DNA methylation and gene expression levels were not present at PD72. In control animals, exercise, but not exercise followed by housing in EC, resulted in higher levels of hippocampal Bdnf gene expression and lower DNA methylation. These studies demonstrate the long-lasting negative impact of developmental alcohol exposure on hippocampal dendritic morphology and support the implementation of exercise and complex environments as therapeutic interventions for individuals with FASD. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 708-725, 2017.

Age-related changes in Egr1 transcription and DNA methylation within the hippocampus.

Aged animals show functional alterations in hippocampal neurons that lead to deficits in synaptic plasticity and changes in cognitive function. Transcription of immediate-early genes (IEGs), including Egr1, is necessary for processes such as long-term potentiation and memory consolidation. Here, we show an age-related reduction in the transcription of Egr1 in the dentate gyrus following spatial behavior, whereas in the area CA1, Egr1 is reduced at rest, but its transcription can be effectively driven by spatial behavior to levels equivalent to those observed in adult animals. One mechanism possibly contributing to these aging-related changes is an age-associated, CpG site-specific change in methylation in DNA associated with the promoter region of the Egr1 gene. Our results add to a growing body of work demonstrating that complex transcriptional and epigenetic changes in the hippocampus significantly contribute to brain and cognitive aging. © 2016 Wiley Periodicals, Inc.

Factors impacting the success of post-mortem sperm rescue in the rhinoceros.

The goal of this study was to identify factors that influenced the ability to successfully rescue sperm post-mortem from rhinoceroses maintained in North American zoos. Factors considered included procedural technicalities, individual rhinoceros characteristics and timing. Gross testicular pathology was noted in 17.4% of males (4/23) but did not impact sperm recovery except in one case of azoospermia (4.3%). Of the males in which sperm recovery was attempted (n=21), 62% yielded quality samples considered adequate for cryopreservation (≥ 30% motility with ≥ 2.0 forward progressive status). A high percentage of males (70.6%; 12/17) from which reproductive tissue was removed an d cooled ≤ 4 h after death yielded quality sperm samples, whereas only 25% (1/4) of males from which tissue was removed>4h after death yielded quality samples. Quality samples were recovered 1-51 h post-mortem from rhinoceroses 8 to 36 years old. Neither type of illness (prolonged or acute), or method of death (euthanasia or natural) affected the ability to harvest quality samples (P > 0.05). The Indian rhinoceros yielded significantly more sperm on average (40 × 10(9)) than the African black rhinoceros (3.6 × 10(9); P < 0.01) and the African white rhinoceros (3.2 × 10(9); P < 0.05). Across all species and samples assessed (n = 11), mean post-thaw sperm motility (41%), was only 15% less than pre-freeze motility (56%) and only decreased to 22% during the 6h post-thaw assessment period. Rhinoceros sperm rescue post-mortem is relatively successful across a wide range of variables, especially when tissues are removed and cooled promptly after death, and should be considered standard practice among zoos.

Effects of developmental alcohol exposure vs. intubation stress on BDNF and TrkB expression in the hippocampus and frontal cortex of neonatal rats.

Third trimester-equivalent alcohol exposure causes significant deficits in hippocampal and cortical neuroplasticity, resulting in alterations to dendritic arborization, hippocampal adult neurogenesis, and performance on learning tasks. The current study investigated the impact of neonatal alcohol exposure (postnatal days 4-9, 5.25 g/kg/day) on expression of brain-derived neurotrophic factor (BDNF) and the tropomyosin-related kinase B (TrkB) receptor in the hippocampal and frontal cortex of infant Long-Evans rats. Levels of BDNF protein were increased in the hippocampus, but not frontal cortex, of alcohol-exposed rats 24h after the last dose, when compared with undisturbed (but not sham-intubated) control animals. BDNF protein levels showed a trend toward increase in hippocampus of sham-intubated animals as well, suggesting an effect of the intubation procedure. TrkB protein was increased in the hippocampus of alcohol-exposed animals compared to sham-intubated pups, indicating an alcohol-specific effect on receptor expression. In addition, expression of bdnf total mRNA in alcohol-exposed and sham-intubated pups was enhanced in the hippocampus; however, there was a differential effect of alcohol and intubation stress on exon I- and IV-specific mRNA transcripts. Further, plasma corticosterone was found to be increased in both alcohol-exposed and sham-intubated pups compared to undisturbed animals. Upregulation of BDNF could potentially represent a neuroprotective mechanism activated following alcohol exposure or stress. The results suggest that alcohol exposure and stress have both overlapping and unique effects on BDNF, and highlight the need for the stress of intubation to be taken into consideration in studies that implement this route of drug delivery.

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.

Neurobiology of secure infant attachment and attachment despite adversity: a mouse model.

Attachment to an abusive caregiver has wide phylogenetic representation, suggesting that animal models are useful in understanding the neural basis underlying this phenomenon and subsequent behavioral outcomes. We previously developed a rat model, in which we use classical conditioning to parallel learning processes evoked during secure attachment (odor-stroke, with stroke mimicking tactile stimulation from the caregiver) or attachment despite adversity (odor-shock, with shock mimicking maltreatment). Here we extend this model to mice. We conditioned infant mice (postnatal day (PN) 7-9 or 13-14) with presentations of peppermint odor and either stroking or shock. We used (14) C 2-deoxyglucose (2-DG) to assess olfactory bulb and amygdala metabolic changes following learning. PN7-9 mice learned to prefer an odor following either odor-stroke or shock conditioning, whereas odor-shock conditioning at PN13-14 resulted in aversion/fear learning. 2-DG data indicated enhanced bulbar activity in PN7-9 preference learning, whereas significant amygdala activity was present following aversion learning at PN13-14. Overall, the mouse results parallel behavioral and neural results in the rat model of attachment, and provide the foundation for the use of transgenic and knockout models to assess the impact of both genetic (biological vulnerabilities) and environmental factors (abusive) on attachment-related behaviors and behavioral development.

Parental responsiveness moderates the association between early-life stress and reduced telomere length.

Early-life stress, such as maltreatment, institutionalization, and exposure to violence, is associated with accelerated telomere shortening. Telomere shortening may thus represent a biomarker of early adversity. Previous studies have suggested that responsive parenting may protect children from the negative biological and behavioral consequences of early adversity. This study examined the role of parental responsiveness in buffering children from telomere shortening following experiences of early-life stress. We found that high-risk children had significantly shorter telomeres than low-risk children, controlling for household income, birth weight, gender, and minority status. Further, parental responsiveness moderated the association between risk and telomere length, with more responsive parenting associated with longer telomeres only among high-risk children. These findings suggest that responsive parenting may have protective benefits on telomere shortening for young children exposed to early-life stress. Therefore, this study has important implications for early parenting interventions.

Neonatal milestones, behavior and growth rate of Sumatran rhinoceros (Dicerorhinus sumatrensis) calves born and bred in captivity.

The birth of Sumatran rhino calves at the Cincinnati Zoo and Botanical Garden offered a unique opportunity to study early development and cow-calf behavior in this elusive, critically endangered species. Study goals were to: (1) record developmental milestones of newborn calves; (2) characterize cow and calf behavior during the first year following birth; (3) compare trends in the behavioral development of a male vs. female calf; and (4) record weight gain and footprint size throughout the first year. In addition to recording eight neonatal milestones, more than 450 hr of behavioral data were collected on each of the two calves (one male and one female) and their dam during the first 12 months of life. Neonatal milestones were achieved within 24 hr of birth with the exception of first defecation, which occurred at 16-18 days. Although nursing bouts decreased slightly in the second half of the year (from once every 90 min to once every 2 hr), they continued to occur frequently throughout the day and night. Therefore, calves grew rapidly from approximately 33 kg at birth to 400 kg at 12 months. Average daily weight gain for the first week was approximately 2.0 kg, whereas average daily weight gain for the remaining 12 months was slight (<1 kg) and did not differ between calves. Eating and resting occupied 70-80% of the Sumatran rhino cow and calves' time and no gender biases in either maternal investment or developmental parameters were noted. Finally, footprint measurements proved valuable for estimating calf age.

Longitudinal fecal hormone analysis for monitoring reproductive activity in the female polar bear (Ursus maritimus).

The objective was to identify suitable enzyme immunoassays to monitor gonadal and placental function in the female polar bear. Immunoreactive progesterone, progesterone metabolite (PdG), estrogen, and androgen metabolite (T) concentrations were measured in fecal samples collected over 24 mo from captive female bears (N = 20). Whereas fecal extracts produced displacement curves parallel to the standard curve for each respective steroid, T and PdG more accurately reflected reproductive events. Concentrations of fecal T increased (P < 0.05) during the breeding season, and brief spikes were associated with estrus and mating. A postovulatory increase in PdG was not always detected, but sustained baseline T after mating appeared consistent with ovulation. Parturient bears excreted higher PdG concentrations (P < 0.05) during expected time of embryo implantation in Fall, and a late gestational rise in fecal T occurred 30 days prepartum. Many nonparturient bears also had a PdG rise in the Fall, suggesting they experienced either pregnancy loss or a pseudopregnancy. Differentiating pregnant and pseudopregnant states was not achieved using fecal PdG alone, but when combined with fecal T, comprehensive diagnoses could be made. Nonparturient bears demonstrated elevated (P < 0.05) fecal T during summer months, whereas parturient bears did not. In summary, noninvasive hormone monitoring techniques were established for the female polar bear. Although this study was directed at facilitating management and breeding efforts of captive polar bears, the methods could be applied to studies of reproductive function in wild populations.

Non-invasive detection of candidate pregnancy protein biomarkers in the feces of captive polar bears (Ursus maritimus).

Currently, there is no method of accurately and non-invasively diagnosing pregnancy in polar bears. Specific proteins may exhibit altered profiles in the feces of pregnant bears, but predicting appropriate candidate proteins to investigate is speculative at best. The objective of this study was to identify potential pregnancy biomarker proteins based on their increased abundance in the feces of pregnant polar bears compared to pseudopregnant females (controls) using two-dimensional in-gel electrophoresis (2D-DIGE) and mass spectrometry (MS). Three 2D-DIGE gels were performed to evaluate fecal protein profiles from controls (n=3) and pregnant polar bears (n=3). There were 2224.67±52.39 (mean±SEM) spots resolved per gel. Of these, only five proteins were elevated in the pregnant group (P<0.05), and seven additional spots tended to be higher (0.0599.9% confidence interval. The 11 spots represented seven distinct proteins, five of which were significantly more abundant in the pregnant group: IgGFc-binding protein, filamin-C, carboxypeptidase B, transthyretin, and immunoglobulin heavy chain variable region. To our knowledge, this was the first study that employed 2D-DIGE to identify differentially expressed proteins in fecal samples to characterize a physiological condition other than those related to gastrointestinal disorders. These promising results provided a strong foundation for ensuing efforts to develop a non-invasive pregnancy assay for use in both captive and wild polar bears.

Factors influencing annual fecal testosterone metabolite profiles in captive male polar bears (Ursus maritimus).

The objectives of this study were to assess the effects of season, breeding activity, age and latitude on fecal testosterone metabolite concentrations in captive, adult male polar bears (Ursus maritimus). Fourteen polar bears from 13 North American zoos were monitored for 12-36 months, producing 25-year-long testosterone profiles. Results indicated that testosterone was significantly higher during the breeding season (early January through the end of May) compared with the non-breeding season with the highest concentrations excreted from early January through late March. Variations in excretion patterns were observed among individuals and also between years within an individual, with testosterone peaks closely associated with breeding activity. Results indicate that fecal testosterone concentrations are influenced by season, breeding activity and age, but not by latitude. This is the first report describing longitudinal fecal testosterone metabolite concentrations in individual adult male polar bears.

Gamete rescue in the African black rhinoceros (Diceros bicornis).

Mortality rates are high among captive African black rhinoceroses (Diceros bicornis), due to increased susceptibility to disease. The ability to rescue genetic material from individuals that die unexpectedly represents a practical approach to assist ex situ conservation efforts. The objectives of the present study were to attempt postmortem oocyte recovery from ovaries of African black rhinoceroses (N = 6) and to test the efficacy of equine protocols for rhinoceros oocyte IVM and IVF using cryopreserved rhinoceros sperm. The interval from ovary removal to oocyte recovery was 25.3 ± 13.9 h (mean ± SD). Ovaries were transported at 4 °C or 22 °C and effects of temperature on postmortem oocyte competence was evaluated. Numbers of oocytes collected per female averaged 15.8 ± 6.9. In total, 95 oocytes were recovered. Of these, 85 were inseminated using homologous sperm and 10 were inseminated using heterologous sperm. Overall, substantial numbers of viable oocytes were retrieved from African black rhinoceros ovaries 1 to 2 days postmortem from ovaries stored at ambient temperature. A proportion of these oocytes matured and underwent penetration and fertilization by heterologous or homologous frozen-thawed rhinoceros sperm. The reproductive competence of postmortem oocytes was further demonstrated by development of a single two-cell embryo. Despite the need for further refinements, gamete rescue in the rhinoceros has promise for producing rhinoceros embryos, as well as testing sperm functions in vitro.

Age-related changes in Arc transcription and DNA methylation within the hippocampus.

The transcription of genes that support memory processes are likely to be impacted by the normal aging process. Because Arc is necessary for memory consolidation and enduring synaptic plasticity, we examined Arc transcription within the aged hippocampus. Here, we report that Arc transcription is reduced within the aged hippocampus compared to the adult hippocampus during both "off line" periods of rest, and following spatial behavior. This reduction is observed within ensembles of CA1 "place cells", which make less mRNA per cell, and in the dentate gyrus (DG) where fewer granule cells are activated by behavior. In addition, we present data suggesting that aberrant changes in methylation of the Arc gene may be responsible for age-related decreases in Arc transcription within CA1 and the DG. Given that Arc is necessary for normal memory function, these subregion-specific epigenetic and transcriptional changes may result in less efficient memory storage and retrieval during aging.

The neurobiology of infant maternal odor learning

Infant rats must learn to identify their mother’s diet-dependent odor. Once learned, maternal odor controls pups’ approach to the mother, their social behavior and nipple attachment. Here we present a review of the research from four different laboratories, which suggests that neural and behavioral responses to the natural maternal odor and neonatal learned odors are similar. Together, these data indicate that pups have a unique learning circuit relying on the olfactory bulb for neural plasticity and on the hyperfunctioning noradrenergic locus coeruleus flooding the olfactory bulb with norepinephrine to support the neural changes. Another important factor making this system unique is the inability of the amygdala to become incorporated into the infant learning circuit. Thus, infant rats appear to be primed in early life to learn odors that will evoke approach responses supporting attachment to the caregiver.