Abnormal reproductive patterns in Przewalski's mares are associated with a loss in gene diversity.

The ex situ population of the Przewalski's horse (Equus ferus przewalskii) is not self-sustaining (20% foaling rate), and the demography is skewed toward aging individuals with low gene diversity. We designed the present study to gain a better understanding of the reproductive biology of the Przewalski's mare and to determine whether age and gene diversity influenced reproductive function. Urine samples were collected 3-7 days/wk from 19 mares from May to September, and ultrasound examinations of follicular structures were performed 3 days/wk for 5 wk from May through July in nine individuals. A high proportion of mares exhibited abnormal (endocrine, 5 [26.3%] of 19; follicular, 2 [22.2%] of 9) or acyclic (endocrine, 4 [21.1%] of 19; follicular, 3 [33.3%] of 9) reproductive patterns. In four cyclic mares, estrous cycle length was 25.1 ± 1.2 days, with 12.2 ± 0.9 days of diestrus. Follicles in cyclic mares grew 1.2 ± 0.6 mm per day and ovulated after reaching 40.4 ± 8.9 mm. Mares with a high coefficient of inbreeding excreted reduced levels of mean urinary estrogens (r(2) = 0.476, P < 0.05), but age had no significant impact on reproductive patterns in this population. Overall, these data suggest that long-term genetic management of this population is necessary to maintain reproductive fitness.

Glucocorticoid response to changes in enclosure size and human proximity in the Persian onager (Equus hemionus onager).

This study investigated glucocorticoid (GC) responses to season and changes in enclosure size and human proximity in the Persian onager (Equus hemionus onager). Enzyme immunoassays were validated to measure GC metabolites in urine and feces (fGCM). Fecal samples were collected from 10 female onagers while in a large pasture, after transport to smaller yards (in greater proximity to people), and 2 months thereafter. Urine samples were collected for 1 year while females were in smaller yards to examine seasonal GC activity. Approximately, 2-fold increases (P < 0.05) were observed in fGCM levels after transport from pasture to yards with increased human exposure, followed by a rapid decline (within -17 days) to baseline (pasture) values. However, responses varied among onagers during the 30 days after translocation, with one female failing to acclimate. Mean fGCM concentrations in smaller yards 2 months after transport were comparable to those in pasture. Seasonal GC concentrations were lowest (P < 0.05) during winter, indicating modest seasonal variability. Results demonstrate an acute increase in GC secretion in Persian onagers that moved from large to small enclosures coincident with increased human activities. Most animals acclimated within 3 weeks, suggesting that this rare equid has retained mechanisms to acclimate to marked alterations in an ex situ environment.

International Society for Wildlife Endocrinology: the future of endocrine measures for reproductive science, animal welfare and conservation biology.

Hormone analysis is a precise and widely accepted tool for monitoring reproductive function and responses to stressors. Although hormones are present and can be measured in various biological matrices, non-invasive methods have gained popularity over the past 30 years as a more practical approach for assessing ovarian, testicular and, more recently, adrenocortical activity in intractable wildlife species. Non-invasive hormone monitoring also has been key to understanding biological mechanisms related to observed behaviours of captive and free-ranging animals. Despite the increasing popularity of this research field, wildlife endocrinologists have not had a specific forum for sharing and discussing their latest findings, technical developments and common challenges. To provide such a communication platform, the International Society for Wildlife Endocrinology (ISWE) was established in 2010, followed by an international meeting held on 3-4 November 2011 at the Toronto Zoo, Canada. Over several sessions, keynote speakers and participants discussed recent developments of new and innovative methods for hormone monitoring, as well as the latest advances in basic endocrinology as applied to adrenal function, reproductive physiology, animal health, ecology and evolution. Here, we introduce ISWE to the scientific community and discuss how this new society will serve as a resource for wildlife endocrinologists worldwide.

A potential link between insulin resistance and iron overload disorder in browsing rhinoceroses investigated through the use of an equine model.

Iron overload disorder afflicts captive rhinoceros but has not been documented in the wild. The specific cause for the disorder has not been identified but is likely associated with diet and management. Compared with wild counterparts, captive rhinoceros eat diets containing more iron, have greater fat stores, and exercise less. It has been suggested that the problem may be linked to development of insulin resistance in the captive population. Given that controlled experiments with sufficient numbers of rhinoceros are logistically not possible, an equine model was used to look for a relationship between iron status and insulin resistance; the nutritional requirements of horses are used as a guide for rhinoceros, because they have similar gastrointestinal tracts. Sixteen horses were tested to determine blood insulin responses to an oral drench of dextrose (0.25 g/kg bodyweight) and a meal of pelleted corn (1.5 g/kg bodyweight). Fasting blood samples were taken 30 and 0 min before administration. Further blood samples were taken every 30 min for 4 hr after administration to determine peak insulin and total area under the insulin curve (AUC). Fasting samples were tested for serum ferritin concentrations. Correlations were determined between ferritin and peak insulin concentrations and insulin AUC after administration of oral dextrose and pelleted corn. The strongest correlation was between ferritin and insulin AUC after dextrose administration (r = 0.61; P = 0.01) followed by AUC after feeding a meal of pelleted corn (r = 0.60; P = 0.01), with the correlation for peak insulin being 0.53 (P = 0.03) after dextrose administration and 0.56 (P = 0.02) after pelleted corn. When evaluating responses by gender, a significant correlation existed only for females, influenced by one insulin resistant individual. These data suggest a potential link between insulin resistance and body stores of iron and also suggest that approaches to reduce the susceptibility to insulin resistance should be incorporated into management of captive browsing rhinoceros.

Effect of body condition, body weight and adiposity on inflammatory cytokine responses in old horses.

Advanced age is associated with a low-grade, systemic inflammatory response characterized by increased inflammatory cytokine production both in vitro and in vivo, termed inflamm-aging. It is also known that increased white adipose tissue, associated with obesity, leads to increased production of inflammatory cytokines. To date, it is unknown whether increased adiposity contributes to the age-related increased inflammatory status. Here we show that peripheral blood mononuclear cells (PBMC) from old horses compared to young horses have increased inflammatory cytokine production; moreover, fat old horses compared to thin old horses have even greater frequencies of lymphocytes and monocytes producing inflammatory cytokines. Therefore, we proposed that decreasing adiposity in old horses would reduce age-associated increases of inflammatory cytokines both in vitro and in vivo, and increasing adiposity in old horses would increase these measurements. To test this hypothesis further, eight old obese horses (20-28 year) were assigned to two consecutive treatments, dietary restriction (DR) during weeks 1-12 and increased dietary intake (DI) during weeks 13-30. Body weight, body condition score (BCS) and percent body fat were measured weekly. PBMC were stimulated in vitro and interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) production was measured by intracellular staining. Levels of nascent IFNgamma and TNFalpha mRNA expression were examined by RT-PCR. Serum concentrations of TNFalpha protein were also measured weekly. Reducing body weight and fat in old horses significantly reduced the percent of IFNgamma and TNFalpha positive lymphocytes and monocytes, and serum levels of TNFalpha protein. Further, when weight and fat increased in these old horses there was a significant increase in inflammatory cytokine production. Regression analysis also revealed significant relationships. These findings demonstrate that age-related obesity potentially plays a role in the dysregulation of inflammatory cytokine production by the immune system with age or inflamm-aging in the horse.

Effects of systemic inflammation on insulin sensitivity in horses and inflammatory cytokine expression in adipose tissue.

OBJECTIVE: To determine whether an inflammatory challenge induces insulin resistance in horses and examine possible contributions of adipose tissue to inflammatory cytokine production. ANIMALS: 15 adult mares. PROCEDURES: Lipopolysaccharide (0.045 mug/kg, IV) or saline solution was administered, and insulin sensitivity was determined by means of the hyperinsulinemic, euglycemic clamp procedure or an adipose tissue biopsy was performed. Adipose tissue samples were collected, and mature adipocytes were obtained. Mature adipocytes were stimulated with lipopolysaccharide or dedifferentiated into preadipocytes and then stimulated with lipopolysaccharide. Interleukin-1, interleukin-6, and tumor necrosis factor A expression in blood, adipose tissue, and adipocytes was quantified with a real-time, reverse transcriptase- PCR assay. RESULTS: Lipopolysaccharide induced a transient increase in insulin sensitivity followed by a reduction in insulin sensitivity at 24 hours. Increased cytokine expression was observed in blood and adipose tissue following administration of lipopolysaccharide, and adipocytes and preadipocytes stimulated with lipopolysaccharide stained positive for tumor necrosis factor A. Expression of interleukin-1, interleukin-6, and tumor necrosis factor A was detected in preadipocytes stimulated with lipopolysaccharide, and interleukin-6 and tumor necrosis factor A were detected in mature adipocytes stimulated with lipopolysaccharide. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that insulin resistance develops following systemic inflammation in horses and suggested that adipose tissue may contribute to this inflammatory response. Methods to regulate insulin sensitivity may improve clinical outcome in critically ill patients.

Rapid phase adjustment of melatonin and core body temperature rhythms following a 6-h advance of the light/dark cycle in the horse.

BACKGROUND: Rapid displacement across multiple time zones results in a conflict between the new cycle of light and dark and the previously entrained program of the internal circadian clock, a phenomenon known as jet lag. In humans, jet lag is often characterized by malaise, appetite loss, fatigue, disturbed sleep and performance deficit, the consequences of which are of particular concern to athletes hoping to perform optimally at an international destination. As a species renowned for its capacity for athletic performance, the consequences of jet lag are also relevant for the horse. However, the duration and severity of jet lag related circadian disruption is presently unknown in this species. We investigated the rates of re-entrainment of serum melatonin and core body temperature (BT) rhythms following an abrupt 6-h phase advance of the LD cycle in the horse. METHODS: Six healthy, 2 yr old mares entrained to a 12 h light/12 h dark (LD 12:12) natural photoperiod were housed in a light-proofed barn under a lighting schedule that mimicked the external LD cycle. Following baseline sampling on Day 0, an advance shift of the LD cycle was accomplished by ending the subsequent dark period 6 h early. Blood sampling for serum melatonin analysis and BT readings were taken at 3-h intervals for 24 h on alternate days for 11 days. Disturbances to the subsequent melatonin and BT 24-h rhythms were assessed using repeated measures ANOVA and analysis of Cosine curve fitting parameters. RESULTS: We demonstrate that the equine melatonin rhythm re-entrains rapidly to a 6-h phase advance of an LD12:12 photocycle. The phase shift in melatonin was fully complete on the first day of the new schedule and rhythm phase and waveform were stable thereafter. In comparison, the advance in the BT rhythm was achieved by the third day, however BT rhythm waveform, especially its mesor, was altered for many days following the LD shift. CONCLUSION: Aside from the temperature rhythm disruption, rapid resynchronization of the melatonin rhythm suggests that the central circadian pacemaker of the horse may possess a particularly robust entrainment response. The consequences for athletic performance remain unknown.

Oral and injectable synthetic progestagens effectively manipulate the estrous cycle in the Przewalski's horse (Equus ferus przewalskii).

To date, there has been limited research on manipulation of the estrous cycle in endangered equids. The objectives of this study were to assess the efficacy of using combinations of: (a) oral altrenogest and PGF2α, and (b) injectable altrenogest and PGF2α for manipulation of ovarian activity in Przewalski's mares. Reproductive cycles were monitored by assessing follicular changes with rectal ultrasound and changes in urinary steroid hormones. In Study 1, five cycling mares were treated with oral altrenogest (n=11 cycles) for 14 days. In Study 2, cycling mares were treated with oral altrenogest for 12 days (n=5 cycles; n=5 mares) or a single injection of biorelease altrenogest (n=10 cycles; n=6 mares). In all study groups, PGF2α was given 2 days before cessation of progestagen treatment. In Study 1, mares responded in six of 11 cycles (54%) where treatment occurred with normal ovarian follicular development post hormone therapy. In Study 2, mares responded in four of five (80%, oral altrenogest) and eight of 10 (80%, injectable altrenogest) cycles with the development of an ovulatory follicle. With the use of injectable altrenogest, there was an obvious suppression of urinary estrogens and progetsagens. These results indicate that manipulation of the estrous cycle of Przewalski's mares can be achieved by administering oral (12 days) or injectable form of altrenogest in conjunction with PGF2α. Findings in the present study may have long term application for the development of timed artificial insemination as a genetic management tool for this critically endangered equid.

Acute systemic inflammation transiently synchronizes clock gene expression in equine peripheral blood.

Peripheral clocks receive timing signals from the master mammalian pacemaker in the suprachiasmatic nucleus (SCN) and function to adaptively anticipate daily changes that influence local physiology. Evidence suggests that peripheral immune activation may act as a resetting signal for circadian clocks in peripheral tissues. We wished to investigate whether acute systemic inflammation could synchronize clock gene expression in equine peripheral blood, a tissue that does not normally oscillate in this species. We report that in vivo administration of lipopolysaccharide (LPS) results in significant upregulation of the core clock genes Per2 and Bmal1 in equine blood, in association with an acute rise in tumor necrosis factor (TNF) alpha and core body temperature compared to vehicle-treated control animals. Furthermore, co-administration of LPS and phenylbutazone, a non-steroidal anti-inflammatory drug (NSAID) known to inhibit prostaglandin (PG) E(2) synthesis in the horse, prevents both the febrile response and the synchronized increase in clock gene expression. However, the rise in Per2 and Bmal1 expression cannot be replicated in equine peripheral blood mononuclear cells (PBMCs) ex vivo by treatment with PGE(2), LPS or a heat shock mimicking the in vivo febrile response. These results may suggest an indirect communication pathway between immune modulators and the molecular machinery of cell clocks in peripheral blood. This potential immune feedback regulation of an equine peripheral clock implies a role for the circadian system in contributing to innate immune reactions and maintaining homeostasis in a tissue that acts as the first line of defense during an infectious challenge.

Evidence of an oscillating peripheral clock in an equine fibroblast cell line and adipose tissue but not in peripheral blood.

The master mammalian pacemaker in the brain controls numerous diverse physiological and behavioral processes throughout the organism. Timing information is continually transmitted from the master clock to peripheral organs to synchronize rhythmic daily oscillations of clock gene transcripts and control local physiology. To investigate the presence of peripheral clocks in the horse, quantitative real-time RT-PCR assays were designed to detect levels of equine clock genes. Expression profiles for Per2, Bmal1 and Cry1 were first determined in a synchronized equine cell line. Subsequently, expression in equine whole blood and adipose tissue was assessed. Robust circadian oscillations of Per2, Bmal1 and Cry1 were observed in vitro. A synchronized molecular clock was also demonstrated in equine adipose tissue although oscillation of Bmal1 was less robust than that of Per2 and Cry1. In contrast to previous studies in humans and rats however, there was no evidence of synchronized clock gene expression in equine peripheral blood. These studies suggest that synchronous control of clock gene oscillation in equine peripheral blood is not as tightly regulated as in other species and may reflect the influence of different evolutionary challenges modifying the function of a peripheral clock.