Successful cryopreservation of African wild dog (Lycaon pictus) spermatozoa: towards developing the frozen zoo

Sperm freezing and artificial insemination can aid species management and conservation of the African wild dog (Lycaon pictus). Freezing attempts have previously been unsuccessful with sperm motility dropping to nearly 0% within 2 h of thawing. We examined the quality of wild dog spermatozoa subjected to 2 routine canine cryopreservation protocols: 1) 1-step dilution in TRIS-egg yolk extender containing 8% glycerol and 20% egg yolk; and 2) 2-step dilution in TRIS-egg yolk extender to a final concentration of 5% glycerol, 20% egg yolk and 0.5% Equex STM. Protocol 2 showed a significantly higher post-thaw viability, acrosome integrity and longevity of spermatozoa with motility present for up to 8 h after thawing; making it suitable for sperm banking and downstream use in species management by artificial insemination for the first time

Social dominance does not affect semen quality in African wild dogs (Lycaon pictus)

Sperm banking and artificial insemination could benefit conservation of endangered African wild dogs (AWD). However, it is not clear whether their strict dominance hierarchy causes subfertility in subdominant males that typically do not breed. Our study investigated the effect of dominance on male reproductive parameters including: faecal glucocorticoids (fGCMs) and androgens (fAMs), testis and prostate volume, preputial gland size, semen collection success, and the number, motility, morphology, viability, acrosome integrity (PSA-FITC), and DNA integrity (TUNEL) of spermatozoa collected by electroejaculation. Samples were obtained from n=12 captive AWDs (4 US packs) in the pre-breeding season and n=28 captive AWDs (n=11 from 4 US packs; n=17 from 3 Namibian packs) in the breeding season. Male hierarchy was clearly determined by behavioural observations in all but 1 Namibian pack. Data were grouped by dominance status and means compared by ANOVA or t-test. P≤0.05 was significant. In the pre-breeding season, there was no significant difference in body weight, fGCMs, fAMs, or prostate and testis volume between dominance groups. Semen was successfully collected from all alphas but only half the subdominants; with urine contamination negatively associated with dominance. Sperm quality was low (17.3 ± 10.2% total motility, 12.8 ± 8.5% progressive motility, 27.4 ± 11.5 x 106 ejaculated spermatozoa, 40.6 ± 9.8% normal morphology, 63.1 ± 5.1% viability, 72.6 ± 5.2% acrosome integrity) with no difference observed in any parameter except progressive motility and normal sperm morphology; which were significantly lower in subdominants (27.7 ± 16.8% vs. 0.0 ± 0.0% and 59.8 ± 13.0% vs. 21.4 ± 5.7%). From pre-breeding to breeding season, testis and prostate volume increased significantly; particularly in beta and gamma males respectively. Prostate volume was higher in alpha than beta males (16.0 ± 6.4 cm3 vs. 5.7 ± 1.4 cm3), but testis volume, body weight, fAMs and fGCMs did not differ between dominance groups (12.0 ± 0.9 cm3, 28.5 ± 0.8 kg, 0.51 ± 0.07 µg/g dry weight - DW, 30.6 ± 2.3 ng/g DW). Semen was successfully collected from 75% of males; with reduced urine contamination. Collection success, urine contamination and preputial gland size were not associated with dominance. Sperm quality improved with significantly greater number, viability, and total motility. However, sperm quality did not differ between dominance groups (47.4 ± 6.7% total motility, 30.5 ± 5.8% progressive motility, 32.3 ± 9.2 x 106 ejaculated spermatozoa, 50.9 ± 5.2% normal morphology, 74.4 ± 4.2% viability, 85.6 ± 3.0% acrosome integrity, 99.7 ± 0.1% DNA integrity). In conclusion, subdominant males are at higher risk of urine contamination and have lower sperm motility and normal morphology when semen is collected in the pre-breeding season. However, their semen is of similar quality to dominant males in the breeding season, indicating that reproductive suppression of subdominant males is only behavioural. Thus, AWD males of all social ranks in the breeding season are suitable candidates for sperm banking

Dog Appeasing Pheromone prevents the testosterone surge, and may reduce contact-dominance and active-submission behaviours after interventions in captive African wild dogs (Lycaon pictus)

African wild dogs (Lycaon pictus) have a complex hierarchical social structure that can lead to aggression; resulting in morbidity and mortality of individuals separated from the pack, or during new pack formation. Aggression and stress might be attenuated by Dog Appeasing Pheromone (DAP). Our placebo-controlled, double-blinded study showed an increase in faecal androgen concentrations within the pack after temporary separation, immobilisation and reintroduction of placebo treated animals, but not for DAP treated individuals. Moreover, DAP treated packs tended to show lower rates of contact-dominance and activesubmission behaviour, but higher rates of non-contact dominance behaviour. Faecal corticosteroid levels rose after intervention in both treatments, probably due to immobilisation-related stress. These preliminary findings suggest that DAP could be a useful management tool to reduce hormones and behaviours potentially leading to aggression in captive African wild dogs

Omega-6 to omega-3 polyunsaturated fatty acid ratio and subsequent mood disorders in young people with at-risk mental states: a 7-year longitudinal study

While cross-sectional studies suggest that patients with mood disorders have a higher ratio of omega-6 to omega-3 polyunsaturated fatty acids (PUFAs) and lower levels of omega-3 PUFAs, it is unknown if a high n-6/3 ratio indicates vulnerability for depression. We tested this hypothesis in a 7-year follow-up study of young individuals with an ultra-high risk (UHR) phenotype. We conducted a secondary analysis of the Vienna omega-3 study, a longitudinal study of omega-3 PUFAs in individuals at UHR for psychosis (n = 69). Levels of n-6 and n-3 PUFAs were measured in the phosphatidylethanolamine fraction of erythrocyte membranes at intake into the study. Mood disorder diagnosis was ascertained with the Structured Clinical Interview for DSM-IV-TR and confirmed by review of medical records and interviews of caregivers. A higher n-6/3 PUFA ratio at baseline predicted mood disorders in UHR individuals over a 7-year (median) follow-up (odds ratio = 1.89, 95% CI = 1.075-3.338, P = 0.03). This association remained significant after adjustment for age, gender, smoking, severity of depressive symptoms at baseline and n-3 supplementation. Consistent results were obtained for individual PUFAs, including lower levels of eicosapentaenoic acid and docosahexaenoic acid. The predictive capacity of these findings was specific to mood disorders as no associations were found for any other psychiatric disorder. To our knowledge, our data provide the first prospective evidence that the n-6/3 PUFA ratio is associated with an increased risk for mood disorders in young people exhibiting an UHR phenotype. These findings may have important implications for treatment and risk stratification beyond clinical characteristics

Stressor- and Corticotropin releasing Factor-induced Reinstatement and Active Stress-related Behavioral Responses are Augmented Following Long-access Cocaine Self-administration by Rats

Rationale Stressful events during periods of drug abstinence likely contribute to relapse in cocaine-dependent individuals. Excessive cocaine use may increase susceptibility to stressor-induced relapse through alterations in brain corticotropin-releasing factor (CRF) responsiveness. Objectives This study examined stressor- and CRF-induced cocaine seeking and other stress-related behaviors in rats with different histories of cocaine self-administration (SA). Materials and methods Rats self-administered cocaine under short-access (ShA; 2 h daily) or long-access (LgA; 6 h daily) conditions for 14 days or were provided access to saline and were tested for reinstatement by a stressor (electric footshock), cocaine or an icv injection of CRF and for behavioral responsiveness on the elevated plus maze, in a novel environment and in the light–dark box after a 14- to 17-day extinction/withdrawal period. Results LgA rats showed escalating patterns of cocaine SA and were more susceptible to reinstatement by cocaine, EFS, or icv CRF than ShA rats. Overall, cocaine SA increased activity in the center field of a novel environment, on the open arms of the elevated plus maze, and in the light compartment of a light–dark box. In most cases, the effects of cocaine SA were dependent on the pattern/amount of cocaine intake with statistically significant differences from saline self-administering controls only observed in LgA rats. Conclusions When examined after several weeks of extinction/ withdrawal, cocaine SA promotes a more active pattern of behavior during times of stress that is associated with a heightened susceptibility to stressor-induced cocaine-seeking behavior and may be the consequence of augmented CRF regulation of addiction-related neurocircuitry

Correction to Severe depression is associated with increased microglial quinolinic acid in subregions of the anterior cingulate gyrus: Evidence for an immune-modulated glutamatergic neurotransmission? [Journal of Neuroinflammation (2013) 10, 34] 10.1186/1742-2094-10-34

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The oxytocin analogue carbetocin prevents emotional impairment and stress-induced reinstatement of opioid-seeking in morphine-abstinent mice.

The main challenge in treating opioid addicts is to maintain abstinence due to the affective consequences associated with withdrawal which may trigger relapse. Emerging evidence suggests a role of the neurohypophysial peptide oxytocin (OT) in the modulation of mood disorders as well as drug addiction. However, its involvement in the emotional consequences of drug abstinence remains unclear. We investigated the effect of 7-day opioid abstinence on the oxytocinergic system and assessed the effect of the OT analogue carbetocin (CBT) on the emotional consequences of opioid abstinence, as well as relapse. Male C57BL/6J mice were treated with a chronic escalating-dose morphine regimen (20-100 mg/kg/day, i.p.). Seven days withdrawal from this administration paradigm induced a decrease of hypothalamic OT levels and a concomitant increase of oxytocin receptor (OTR) binding in the lateral septum and amygdala. Although no physical withdrawal symptoms or alterations in the plasma corticosterone levels were observed after 7 days of abstinence, mice exhibited increased anxiety-like and depressive-like behaviors and impaired sociability. CBT (6.4 mg/kg, i.p.) attenuated the observed negative emotional consequences of opioid withdrawal. Furthermore, in the conditioned place preference paradigm with 10 mg/kg morphine conditioning, CBT (6.4 mg/kg, i.p.) was able to prevent the stress-induced reinstatement to morphine-seeking following extinction. Overall, our results suggest that alterations of the oxytocinergic system contribute to the mechanisms underlying anxiety, depression, and social deficits observed during opioid abstinence. This study also highlights the oxytocinergic system as a target for developing pharmacotherapy for the treatment of emotional impairment associated with abstinence and thereby prevention of relapse

Corticotropin Releasing Factor-Induced CREB Activation in Striatal Neurons Occurs via a Novel Gβγ Signaling Pathway

The peptide corticotropin-releasing factor (CRF) was initially identified as a critical component of the stress response. CRF exerts its cellular effects by binding to one of two cognate G-protein coupled receptors (GPCRs), CRF receptor 1 (CRFR1) or 2 (CRFR2). While these GPCRs were originally characterized as being coupled to Gαs, leading to downstream activation of adenylyl cyclase (AC) and subsequent increases in cAMP, it has since become clear that CRFRs couple to and activate numerous other downstream signaling cascades. In addition, CRF signaling influences the activity of many diverse brain regions, affecting a variety of behaviors. One of these regions is the striatum, including the nucleus accumbens (NAc). CRF exerts profound effects on striatal-dependent behaviors such as drug addiction, pair-bonding, and natural reward. Recent data indicate that at least some of these behaviors regulated by CRF are mediated through CRF activation of the transcription factor CREB. Thus, we aimed to elucidate the signaling pathway by which CRF activates CREB in striatal neurons. Here we describe a novel neuronal signaling pathway whereby CRF leads to a rapid Gβγ- and MEK-dependent increase in CREB phosphorylation. These data are the first descriptions of CRF leading to activation of a Gβγ-dependent signaling pathway in neurons, as well as the first description of Gβγ activation leading to downstream CREB phosphorylation in any cellular system. Additionally, these data provide additional insight into the mechanisms by which CRF can regulate neuronal function

Stress-Induced Reinstatement of Drug Seeking: 20 Years of Progress

In human addicts, drug relapse and craving are often provoked by stress. Since 1995, this clinical scenario has been studied using a rat model of stress-induced reinstatement of drug seeking. Here, we first discuss the generality of stress-induced reinstatement to different drugs of abuse, different stressors, and different behavioral procedures. We also discuss neuropharmacological mechanisms, and brain areas and circuits controlling stress-induced reinstatement of drug seeking. We conclude by discussing results from translational human laboratory studies and clinical trials that were inspired by results from rat studies on stress-induced reinstatement. Our main conclusions are (1) The phenomenon of stress-induced reinstatement, first shown with an intermittent footshock stressor in rats trained to self-administer heroin, generalizes to other abused drugs, including cocaine, methamphetamine, nicotine, and alcohol, and is also observed in the conditioned place preference model in rats and mice. This phenomenon, however, is stressor specific and not all stressors induce reinstatement of drug seeking. (2) Neuropharmacological studies indicate the involvement of corticotropin-releasing factor (CRF), noradrenaline, dopamine, glutamate, kappa/dynorphin, and several other peptide and neurotransmitter systems in stress-induced reinstatement. Neuropharmacology and circuitry studies indicate the involvement of CRF and noradrenaline transmission in bed nucleus of stria terminalis and central amygdala, and dopamine, CRF, kappa/dynorphin, and glutamate transmission in other components of the mesocorticolimbic dopamine system (ventral tegmental area, medial prefrontal cortex, orbitofrontal cortex, and nucleus accumbens). (3) Translational human laboratory studies and a recent clinical trial study show the efficacy of alpha-2 adrenoceptor agonists in decreasing stress-induced drug craving and stress-induced initial heroin lapse

Ultradian Cortisol Pulsatility Encodes a Distinct, Biologically Important Signal

Cortisol is released in ultradian pulses. The biological relevance of the resulting fluctuating cortisol concentration has not been explored.Determination of the biological consequences of ultradian cortisol pulsatility.A novel flow through cell culture system was developed to deliver ultradian pulsed or continuous cortisol to cells. The effects of cortisol dynamics on cell proliferation and survival, and on gene expression were determined. In addition, effects on glucocorticoid receptor (GR) expression levels and phosphorylation, as a potential mediator, were measured.Pulsatile cortisol caused a significant reduction in cell survival compared to continuous exposure of the same cumulative dose, due to increased apoptosis. Comprehensive analysis of the transcriptome response by microarray identified genes with a differential response to pulsatile versus continuous glucocorticoid delivery. These were confirmed with qRT-PCR. Several transcription factor binding sites were enriched in these differentially regulated target genes, including CCAAT-displacement protein (CDP). A CDP regulated reporter gene (MMTV-luc) was, as predicted, also differentially regulated by pulsatile compared to continuous cortisol delivery. Importantly there was no effect of cortisol delivery kinetics on either GR expression, or activation (GR phosphoSer(211)).Cortisol oscillations exert important effects on target cell gene expression, and phenotype. This is not due to differences in cumulative cortisol exposure, or either expression, or activation of the GR. This suggests a novel means to regulate GR function