Retinal Tissue Shows Glial Changes in a Dravet Syndrome Knock-in Mouse Model.

Dravet syndrome (DS) is an epileptic encephalopathy caused by mutations in the Scn1a gene encoding the α1 subunit of the Nav1.1 sodium channel, which is associated with recurrent and generalized seizures, even leading to death. In experimental models of DS, histological alterations have been found in the brain; however, the retina is a projection of the brain and there are no studies that analyze the possible histological changes that may occur in the disease. This study analyzes the retinal histological changes in glial cells (microglia and astrocytes), retinal ganglion cells (RGCs) and GABAergic amacrine cells in an experimental model of DS (Syn-Cre/Scn1aWT/A1783V) compared to a control group at postnatal day (PND) 25. Retinal whole-mounts were labeled with anti-GFAP, anti-Iba-1, anti-Brn3a and anti-GAD65/67. Signs of microglial and astroglial activation, and the number of Brn3a+ and GAD65+67+ cells were quantified. We found retinal activation of astroglial and microglial cells but not death of RGCs and GABAergic amacrine cells. These changes are similar to those found at the level of the hippocampus in the same experimental model in PND25, indicating a relationship between brain and retinal changes in DS. This suggests that the retina could serve as a possible biomarker in DS.

Modeling Parkinson's Disease Neuropathology and Symptoms by Intranigral Inoculation of Preformed Human α-Synuclein Oligomers.

The accumulation of aggregated α-synuclein (αSyn) is a hallmark of Parkinson's disease (PD). Current evidence indicates that small soluble αSyn oligomers (αSynOs) are the most toxic species among the forms of αSyn aggregates, and that size and topological structural properties are crucial factors for αSynOs-mediated toxicity, involving the interaction with either neurons or glial cells. We previously characterized a human αSynO (H-αSynO) with specific structural properties promoting toxicity against neuronal membranes. Here, we tested the neurotoxic potential of these H-αSynOs in vivo, in relation to the neuropathological and symptomatic features of PD. The H-αSynOs were unilaterally infused into the rat substantia nigra pars compacta (SNpc). Phosphorylated αSyn (p129-αSyn), reactive microglia, and cytokine levels were measured at progressive time points. Additionally, a phagocytosis assay in vitro was performed after microglia pre-exposure to αsynOs. Dopaminergic loss, motor, and cognitive performances were assessed. H-αSynOs triggered p129-αSyn deposition in SNpc neurons and microglia and spread to the striatum. Early and persistent neuroinflammatory responses were induced in the SNpc. In vitro, H-αSynOs inhibited the phagocytic function of microglia. H-αsynOs-infused rats displayed early mitochondrial loss and abnormalities in SNpc neurons, followed by a gradual nigrostriatal dopaminergic loss, associated with motor and cognitive impairment. The intracerebral inoculation of structurally characterized H-αSynOs provides a model of progressive PD neuropathology in rats, which will be helpful for testing neuroprotective therapies.

Brain activity of anandamide: a rewarding bliss?

Anandamide is a lipid mediator that acts as an endogenous ligand of CB1 receptors. These receptors are also the primary molecular target responsible for the pharmacological effects of Δ9-tetrahydrocannabinol, the psychoactive ingredient in Cannabis sativa. Several studies demonstrate that anandamide exerts an overall modulatory effect on the brain reward circuitry. Several reports suggest its involvement in the addiction-producing actions of other abused drugs, and it can also act as a behavioral reinforcer in animal models of drug abuse. Importantly, all these effects of anandamide appear to be potentiated by pharmacological inhibition of its metabolic degradation. Enhanced brain levels of anandamide after treatment with inhibitors of fatty acid amide hydrolase, the main enzyme responsible for its degradation, seem to affect the rewarding and reinforcing actions of many drugs of abuse. In this review, we will provide an overview from a preclinical perspective of the current state of knowledge regarding the behavioral pharmacology of anandamide, with a particular emphasis on its motivational/reinforcing properties. We will also discuss how modulation of anandamide levels through inhibition of enzymatic metabolic pathways could provide a basis for developing new pharmaco-therapeutic tools for the treatment of substance use disorders.

Impaired brain endocannabinoid tone in the activity-based model of anorexia nervosa.

OBJECTIVE: Despite the growing knowledge on the functional relationship between an altered endocannabinoid (eCB) system and development of anorexia nervosa (AN), to date no studies have investigated the central eCB tone in the activity-based anorexia (ABA) model that reproduces key aspects of human AN. METHOD: We measured levels of two major eCBs, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), those of two eCB-related lipids, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and the cannabinoid type-1 receptor (CB1R) density in the brain of female ABA rats, focusing on areas involved in homeostatic and rewarding-related regulation of feeding behavior (i.e., prefrontal cortex, nucleus accumbens, caudato putamen, amygdala, hippocampus and hypothalamus). Analysis was carried out also at the end of recovery from the ABA condition. RESULTS: At the end of the ABA induction phase, 2-AG was significantly decreased in ABA rats in different brain areas but not in the caudato putamen. No changes were detected in AEA levels in any region, whereas the levels of OEA and PEA were decreased exclusively in the hippocampus and hypothalamus. Furthermore, CB1R density was decreased in the dentate gyrus of hippocampus and in the lateral hypothalamus. After recovery, both 2-AG levels and CB1R density were partially normalized in some areas. In contrast, AEA levels became markedly reduced in all the analyzed areas. DISCUSSION: These data demonstrate an altered brain eCB tone in ABA rats, further supporting the involvement of an impaired eCB system in AN pathophysiology that may contribute to the maintenance of some symptomatic aspects of the disease.

Novel sulfenamides and sulfonamides based on pyridazinone and pyridazine scaffolds as CB1 receptor ligand antagonists.

A series of sulfenamide and sulfonamide derivatives was synthesized and evaluated for the affinity at CB1 and CB2 receptors. The N-bornyl-S-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide, compound 11, displayed good affinity and high selectivity for CB1 receptors (Ki values of 44.6 nM for CB1 receptors and >40 µM for CB2 receptors, respectively). The N-isopinocampheyl-sulfenamide 12 and its sulfonamide analogue 22 showed similar selectivity for CB1 receptors with Ki values of 75.5 and 73.2 nM, respectively. These novel compounds behave as antagonists/inverse agonists at CB1 receptor in the [35S]-GTPγS binding assays, and none showed adequate predictive blood-brain barrier permeation, exhibiting low estimated LD50. However, testing compound 12 in a supraspinal analgesic test (hot-plate) revealed that it was as effective as the classic CB1 receptor antagonist rimonabant, in reversing the analgesic effect of a cannabinoid agonist.

Disease-modifying effects of cannabidiol, ß-caryophyllene and their combination in Syn1-Cre/Scn1aWT/A1783V mice, a preclinical model of Dravet syndrome.

Cannabidiol (CBD) has been recently approved as an antiseizure agent in Dravet Syndrome (DS), a pediatric epileptic encephalopathy, but CBD could also be active against associated comorbidities. Such associated comorbidities were also attenuated by the sesquiterpene ß-caryophyllene (BCP). Here, we have compared the efficacy of both compounds and further initiated the analysis of a possible additive effect between both compounds in relation with these comorbidities using two experimental approaches. The first experiment was aimed at comparing the benefits of CBD and BCP, including their combination in conditional knock-in Scn1a-A1783V mice, an experimental model of DS, treated since the postnatal day 10th to 24th. As expected, DS mice showed impairment in limb clasping, delay in the appearance of hindlimb grasp reflex and additional behavioural disturbances (e.g., hyperactivity, cognitive deterioration, social interaction deficits). This behavioural impairment was associated with marked astroglial and microglial reactivities in the prefrontal cortex and the hippocampal dentate gyrus. BCP and CBD administered alone were both able to partially attenuate the behavioural disturbances and the glial reactivities, with apparently greater efficacy against glial reactivities obtained with BCP, whereas superior effects in a few specific parameters were obtained when both compounds were combined. In the second experiment, we investigated this additive effect in cultured BV2 cells treated with BCP and/or CBD and stimulated with LPS. As expected, addition of LPS induced a marked increase in several inflammation-related markers (e.g., TLR4, COX-2, iNOS, catalase, TNF-α, IL-1ß), as well as elevated Iba-1 immunostaining. Treatment with BCP or CBD attenuated these elevations, but, again and in general, superior results were obtained when both cannabinoids were combined. In conclusion, our results support the interest to continue investigating the combination of BCP and CBD to improve the therapeutic management of DS in relation with their disease-modifying properties.

Endocannabinoid-Binding Receptors as Drug Targets.

Cannabis plant has been used from ancient times with therapeutic purposes for treating human pathologies, but the identification of the cellular and molecular mechanisms underlying the therapeutic properties of the phytocannabinoids, the active compounds in this plant, occurred in the last years of the past century. In the late 1980s and early 1990s, seminal studies demonstrated the existence of cannabinoid receptors and other elements of the so-called endocannabinoid system. These G protein-coupled receptors (GPCRs) are a key element in the functions assigned to endocannabinoids and appear to serve as promising pharmacological targets. They include CB1, CB2, and GPR55, but also non-GPCRs can be activated by endocannabinoids, like ionotropic receptor TRPV1 and even nuclear receptors of the PPAR family. Their activation, inhibition, or simply modulation have been associated with numerous physiological effects at both central and peripheral levels, which may have therapeutic value in different human pathologies, then providing a solid experimental explanation for both the ancient medicinal uses of Cannabis plant and the recent advances in the development of cannabinoid-based specific therapies. This chapter will review the scientific knowledge generated in the last years around the research on the different endocannabinoid-binding receptors and their signaling mechanisms. Our intention is that this knowledge may help readers to understand the relevance of these receptors in health and disease conditions, as well as it may serve as the theoretical basis for the different experimental protocols to investigate these receptors and their signaling mechanisms that will be described in the following chapters.

Adaptive Response to Gillnets Bycatch in a North Sardinia Mediterranean Shag (Gulosus aristotelis desmarestii) Population.

Mediterranean Shag (Gulosus aristotelis desmarestii) is a seabird endemic to the Mediterranean and Black Seas, recently included in the IUCN list of threatened Species. Most of the reproductive colonies are hosted in Sardinia and surrounding islets. Bycatch in fishing nets is one of the most significant threats for this population. Our work aimed to assess alterations in the sex ratio caused by bycatch and to study the adaptive response of the population to a skewed adult sex ratio. The sex ratio of Mediterranean Shags found drowned in the gillnets near the colonies and that of the nestlings of the Corcelli (northeast Sardinia) colony was determined using the sex-linked polymorphism of the gene Chromobox-Helicase-DNA-binding 1. The data of the shags found drowned in gillnets evidenced a high mortality rate (83.3%; p < 0.001) and a larger size of males (35% heavier than females, p < 0.05) compared to females, supporting the theory that heavier individuals are able to forage at great depths. With 64.8% of the nestlings being male, the sex ratio of nestlings was statistically different from parity (p < 0.05). Furthermore, it was related to the brood size. In one- and two-chick broods, 73% and 70% of nestlings, respectively, were males, while in three-chick broods, only 33% were males. Our data identify the higher rate of male shags drowned in gillnets as a factor causing an alteration of the sex ratio in the Mediterranean Shag population. According to the Sex Allocation Theory, an adaptive adjustment of sex made by adult females restores the Mendelian sex ratio in the population.

Preclinical investigation of ß-caryophyllene as a therapeutic agent in an experimental murine model of Dravet syndrome.

Dravet Syndrome (DS) is caused by mutations in the Scn1a gene encoding the α1 subunit of the sodium channel Nav1.1, which results in febrile seizures that progress to severe tonic-clonic seizures and associated comorbidities. Treatment with cannabidiol has been approved for the management of seizures in DS patients, but it appears to be also active against associated comorbidities. In this new study, we have investigated ß-caryophyllene (BCP), a cannabinoid with terpene structure that appears to also have a broad-spectrum profile, as a useful therapy against both seizuring activity and progression of associated comorbidities. This has been studied in heterozygous conditional knock-in mice carrying a missense mutation (A1783V) in Scn1a gene expressed exclusively in neurons of the Central Nervous System (Syn-Cre/Scn1aWT/A1783V), using two experimental approaches. In the first approach, an acute treatment with BCP was effective against seizuring activity induced by pentylenetetrazole (PTZ) in wildtype (Scn1aWT/WT) and also in Syn-Cre/Scn1aWT/A1783V mice, with these last animals having a greater susceptibility to PTZ. Such benefits were paralleled by a BCP-induced reduction in PTZ-induced reactive astrogliosis (labelled with GFAP) and microgliosis (labelled with Iba-1) in the prefrontal cortex and the hippocampal dentate gyrus, which were visible in both wildtype (Scn1aWT/WT) and Syn-Cre/Scn1aWT/A1783V mice. In the second approach, both genotypes were treated repeatedly with BCP to investigate its effects on several DS comorbidities. Thus, BCP corrected important behavioural abnormalities of Syn-Cre/Scn1aWT/A1783V mice (e.g. delayed appearance of hindlimb grasp reflex, induction of clasping response, motor hyperactivity, altered social interaction and memory impairment), attenuated weight loss, and slightly delayed premature mortality. Again, these benefits were paralleled by a BCP-induced reduction in reactive astrogliosis and microgliosis in the prefrontal cortex and the hippocampal dentate gyrus typical of Syn-Cre/Scn1aWT/A1783V mice. In conclusion, BCP was active in Syn-Cre/Scn1aWT/A1783V mice against seizuring activity (acute treatment) and against several comorbidities (repeated treatment), in both cases in association with its capability to reduce glial reactivity in areas related to these behavioural abnormalities. This situates BCP in a promising position for further preclinical evaluation towards a close translation to DS patients.

Neuroprotective effects of phytocannabinoid-based medicines in experimental models of Huntington's disease.

We studied whether combinations of botanical extracts enriched in either Δ(9)-tetrahydrocannabinol (Δ(9)-THC) or cannabidiol (CBD), which are the main constituents of the cannabis-based medicine Sativex, provide neuroprotection in rat models of Huntington's disease (HD). We used rats intoxicated with 3-nitropropionate (3NP) that were given combinations of Δ(9)-THC- and CBD-enriched botanical extracts. The issue was also studied in malonate-lesioned rats. The administration of Δ(9)-THC- and CBD-enriched botanical extracts combined in a ratio of 1:1 as in Sativex attenuated 3NP-induced GABA deficiency, loss of Nissl-stained neurons, down-regulation of CB(1) receptor and IGF-1 expression, and up-regulation of calpain expression, whereas it completely reversed the reduction in superoxide dismutase-1 expression. Similar responses were generally found with other combinations of Δ(9)-THC- and CBD-enriched botanical extracts, suggesting that these effects are probably related to the antioxidant and CB(1) and CB(2) receptor-independent properties of both phytocannabinoids. In fact, selective antagonists for both receptor types, i.e., SR141716 and AM630, respectively, were unable to prevent the positive effects on calpain expression caused in 3NP-intoxicated rats by the 1:1 combination of Δ(9)-THC and CBD. Finally, this combination also reversed the up-regulation of proinflammatory markers such as inducible nitric oxide synthase observed in malonate-lesioned rats. In conclusion, this study provides preclinical evidence in support of a beneficial effect of the cannabis-based medicine Sativex as a neuroprotective agent capable of delaying disease progression in HD, a disorder that is currently poorly managed in the clinic, prompting an urgent need for clinical trials with agents showing positive results in preclinical studies.

Melatonin protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner.

Cryopreservation harms spermatozoa at different levels and thus impairs their fertilizing ability. The role of melatonin in protecting spermatozoa from different kind injuries has been widely reported. Thus, this study tested whether the addition of melatonin to ram semen freezing extender could exert a protective effect and ameliorate postthawing sperm function. Melatonin was added to recommended ram extender to yield five different final concentrations: 0.001, 0.01, 0.1, 1, and 10 mm. A control group without melatonin supplementation was included. Spermatozoa viability, motility parameters, and intracellular ATP concentrations were evaluated both before and after cryopreservation, while DNA integrity and in vitro fertilizing ability were evaluated only after thawing. Obtained results showed that the concentration of 1 mm melatonin led to higher viability rates, higher percentages of total motile and progressive motile spermatozoa, higher percentages of spermatozoa with average rapid and medium velocity, higher intracellular ATP concentrations, and higher DNA integrity among semen frozen in control and melatonin-supplemented extenders (P<0.05). In addition, results obtained after the IVF test showed that at 1 mm concentration, melatonin led to a faster first embryonic division and to higher total cleavage rates compared to the other experimental groups (P<0.05). No difference in embryo output was observed among the six experimental groups. In conclusion, the addition of melatonin to ram semen freezing extender protected spermatozoa during cryopreservation in a dose-dependent manner. These results are likely to be mediated by its well-known antioxidant properties, even if a direct action of the indolamine cannot be ruled out.

Genetic characterization and implications for conservation of the last autochthonous Mouflon population in Europe.

Population genetic studies provide accurate information on population structure, connectivity, and hybridization. These are key elements to identify units for conservation and define wildlife management strategies aimed to maintain and restore biodiversity. The Mediterranean island of Sardinia hosts one of the last autochthonous mouflon populations, descending from the wild Neolithic ancestor. The first mouflon arrived in Sardinia ~ 7000 years ago and thrived across the island until the twentieth century, when anthropogenic factors led to population fragmentation. We analysed the three main allopatric Sardinian mouflon sub-populations, namely: the native sub-populations of Montes Forest and Mount Tonneri, and the reintroduced sub-population of Mount Lerno. We investigated the spatial genetic structure of the Sardinian mouflon based on the parallel analysis of 14 highly polymorphic microsatellite loci and mitochondrial D-loop sequences. The Montes Forest sub-population was found to harbour the ancestral haplotype in the phylogeny of European mouflon. We detected high levels of relatedness in all the sub-populations and a mitochondrial signature of hybridization between the Mount Lerno sub-population and domestic sheep. Our findings provide useful insights to protect such an invaluable genetic heritage from the risk of genetic depletion by promoting controlled inter-population exchange and drawing informed repopulation plans sourcing from genetically pure mouflon stocks.

Neuropathological Characterization of a Dravet Syndrome Knock-In Mouse Model Useful for Investigating Cannabinoid Treatments.

Dravet syndrome (DS) is an epileptic syndrome caused by mutations in the Scn1a gene encoding the α1 subunit of the sodium channel Nav1.1, which is associated with febrile seizures that progress to severe tonic-clonic seizures and associated comorbidities. Treatment with cannabidiol has been approved to reduce seizures in DS, but it may also be active against these comorbidities. The aim of this study was to validate a new mouse model of DS having lower mortality than previous models, which may serve to further evaluate therapies for the long-term comorbidities. This new model consists of heterozygous conditional knock-in mice carrying a missense mutation (A1783V) in Scn1a gene expressed exclusively in neurons of the CNS (Syn-Cre/Scn1aWT/A1783V). These mice have been used here to determine the extent and persistence of the behavioral deterioration in different postnatal days (PND), as well as to investigate the alterations that the disease produces in the endocannabinoid system and the contribution of inflammatory events and impaired neurogenesis in the pathology. Syn-Cre/Scn1aWT/A1783V mice showed a strong reduction in hindlimb grasp reflex at PND10, whereas at PND25, they presented spontaneous convulsions and a greater susceptibility to pentylenetetrazole-induced seizures, marked hyperactivity, deficient spatial working memory, lower levels of anxiety, and altered social interaction behavior. These differences disappeared at PND40 and PND60, except the changes in social interaction and anxiety. The analysis of CNS structures associated with these behavioral alterations revealed an elevated glial reactivity in the prefrontal cortex and the dentate gyrus. This was associated in the dentate gyrus with a greater cell proliferation detected with Ki67 immunostaining, whereas double-labeling analyses identified that proliferating cells were GFAP-positive suggesting failed neurogenesis but astrocyte proliferation. The analysis of the endocannabinoid system of Syn-Cre/Scn1aWT/A1783V mice confirmed reductions in CB1 receptors and MAGL and FAAH enzymes, mainly in the cerebellum but also in other areas, whereas CB2 receptors became upregulated in the hippocampus. In conclusion, Syn-Cre/Scn1aWT/A1783V mice showed seizuring susceptibility and several comorbidities (hyperactivity, memory impairment, less anxiety, and altered social behavior), which exhibited a pattern of age expression similar to DS patients. Syn-Cre/Scn1aWT/A1783V mice also exhibited greater glial reactivity and a reactive response in the neurogenic niche, and regional changes in the status of the endocannabinoid signaling, events that could contribute in behavioral impairment.

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model.

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation.

Ejaculate collection efficiency and post-thaw semen quality in wild-caught Griffon vultures from the Sardinian population.

This study aimed to test the feasibility of a programme of semen collection and cryopreservation in Griffon vultures. Four wild-caught individuals kept in captivity because of unrecoverable traumas were used. Semen collection attempts were made twice a week during three consecutive reproductive seasons (December - March) using the abdominal massage method. Ejaculation was successfully induced between late January and late February. Semen collection efficiency was rather low (27.9%) and it did not vary among individuals (p > 0.05). No differences were found in ejaculate volumes (12.5 +/- 9.1 microl), spermatozoa concentration (28.4 +/- 30.9 million cells/ml) and viability (61.3 +/- 13.9%) among the 4 vultures. ATP values differed among the four vultures (p < 0.001); B showed higher nucleotide concentration than both C and D, while it did not differ form A, whose values were higher compared with D. After freezing and thawing, semen in vitro viability, DNA integrity and ATP intracellular concentration were determined. Spermatozoa viability after thawing did not differ among the four individuals (52.6 +/- 5.8 in A, 53.4 +/- 4.6 in B, 50.4 +/- 3.2 in C, 42.5 +/- 2.7 in D), but it decreased significantly compared to fresh semen (p < 0.05). During 4 hrs in vitro culture, spermatozoa collected from B maintained over time a higher viability in vitro when compared to A, C and D. As evaluated by the comet assay method, DNA fragmentation after freezing and thawing did not differ in the 4 vultures. ATP concentration in frozen/thawed semen was significantly lower than in fresh semen (p < 0.0001). This study indicates that semen cryopreservation can be considered as a useful tool in the conservation of Griffon vulture genetic resources, but further studies are needed to optimize this technique.

Exogenous melatonin positively influences follicular dynamics, oocyte developmental competence and blastocyst output in a goat model.

The role of melatonin in modulating mammalian reproduction is of particular interest; however, its effects on ovarian follicles and their oocytes still remain to be characterized. This study determined the influence of melatonin treatment on follicular growth patterns and on in vitro oocyte developmental competence. In a first experiment, the effects of melatonin supplementation on follicular dynamics were evaluated using daily transrectal ultrasonographies for 21 days, in 7 multiparous Sarda goats receiving a subcutaneous implant of 18 mg of melatonin and in 5 control untreated does. Melatonin caused more follicular waves (5.2 +/- 0.2 versus 4 +/- 0.3; P < 0.05) as the waves were shortened at around 2 days when compared with the non-melatonin treated control goats (P < 0.001). Oocyte developmental competence was evaluated in a second experiment by applying procedures for in vitro embryo production. There were no significant differences in the total number of oocytes obtained from 6 control (n = 192) and 7 melatonin-treated (n = 265) goats given follicle stimulating hormone to induce follicular development. Differences in oocyte developmental competence between the two groups became evident after in vitro fertilization and culture; melatonin increased the rate of cleaved oocytes in comparison with control animals (82.5 versus 63.4%; P < 0.001), advanced timing of embryo development and enhanced blastocyst output (31.5 versus 16.3%; P < 0.01). However, blastocyst quality, as evaluated by cryotolerance and gene expression analysis, was not found to be different between the groups. In conclusion, in vivo melatonin treatment is beneficial for increasing ovarian follicle turnover and improving oocyte developmental competence and kinetics of the blastocyst.

In vitro production and cryotolerance of prepubertal and adult goat blastocysts obtained from oocytes collected by laparoscopic oocyte-pick-up (LOPU) after FSH treatment.

This study compares the developmental capacity and cryotolerance of embryos produced from oocytes of stimulated prepubertal and adult Sarda goats. Twelve prepubertal and 13 adult goats were each given 110 and 175 IU FSH, respectively, and cumulus-oocyte complexes (COCs) were collected by laparoscopic oocyte-pick-up (LOPU). After in vitro maturation, fertilisation and culture (IVMFC), blastocysts were vitrified, warmed and blastocoel re-expansion and gene expression were evaluated. Prepubertal goats produced a higher COCs number than adults (mean +/- s.e.m., 89.67 +/- 5.74 and 26.69 +/- 3.66, respectively; P < 0.01). Lower developmental competence was demonstrated in the prepubertal oocytes as shown by a higher number of COCs discarded before IVM (21.1% and 14.7% for prepubertals and adults, respectively; P < 0.01) and IVF (23.4% v. 9.1%; P < 0.01) and by the lower cleavage (55.6% and 70.3%, respectively; P < 0.01) and blastocyst rates (24.2% and 33.9%, respectively; P < 0.05). Compared with the adult, prepubertal vitrified/warmed blastocysts showed significantly (P < 0.05) lower in vitro viability, as determined by the re-expansion rate (62.5% and 40.3%). No differences were observed in the time required for blastocoel re-expansion or in cyclin B1, E-cadherin, Na/K ATPase, HSP90beta and aquaporin 3 messenger RNA quantity. These results show that in vitro-produced embryos produced from prepubertal goat oocytes have a lower developmental rate and cryotolerance compared with their adult counterparts. However, we can assume that the quality of re-expanded embryos does not differ between the two groups.

Animal Models of Eating Disorders.

Eating disorders (EDs) include a range of chronic and disabling pathologies characterized by persistent maladaptive eating habits and/or behaviors aimed at controlling body shape and size, with important consequences on physical health. Different animal models of EDs have been developed to investigate pharmacological, environmental, and genetic determinants that contribute to the development and maintenance of these disorders as well as for the identification of potential therapeutic targets. In this chapter, we will provide an overview of the most useful animal models of EDs, focusing mainly on those used to study anorexia nervosa and binge eating disorder.

Identification of an ancestral haplotype in the mitochondrial phylogeny of the ovine haplogroup B.

BACKGROUND: European mouflon (Ovis orientalis musimon) has been reintroduced in mainland Europe since the 18th-century sourcing from the Sardinian and Corsican autochthonous mouflon populations. The European mouflon is currently considered the feral descendent of the Asian mouflon (O. orientalis), and the result of first wave of sheep domestication occurred 11,000 years ago in the Fertile Crescent, and brought to Corsica and Sardinia ca. 6,000 years ago, where they still live as autochthonous populations. However, this phylogeny is based on mitogenome sequences of European mouflon individuals exclusively. METHODS: We sequenced the first complete mtDNA of the long-time isolated Sardinian mouflon and compared it with several ovine homologous sequences, including mouflon from mainland Europe and samples representative of the five known mitochondrial domestic sheep haplogroups. We applied Bayesian inference, Maximum Likelihood and Integer Neighbour-Joining network methods and provided a robust, fully-resolved phylogeny with strong statistical support for all nodes. RESULTS: We identified an early split (110,000 years ago) of the Sardinian mouflon haplotype from both sheep and mainland European mouflon belonging to haplogroup B, the latter two sharing a more recent common ancestor (80,000 years ago). Further, the Sardinian mouflon sequence we generated had the largest genetic distance from domestic sheep haplogroups (0.0136 ± 0.004) among mouflon species. Our results suggest the Sardinian mouflon haplotype as the most ancestral in the HPG-B lineage, hence partially redrawing the known phylogeny of the genus Ovis.

Mitochondrial D-loop Sequence Variability in Three Native Insular Griffon Vulture (Gyps fulvus) Populations from the Mediterranean Basin.

The islands of Sardinia, Crete, and Cyprus are hosting the last native insular griffon populations in the Mediterranean basin. Their states have been evaluated from "vulnerable" to "critically endangered". The sequence analysis of molecular markers, particularly the mtDNA D-loop region, provides useful information in studying the evolution of closely related taxa and the conservation of endangered species. Therefore, a study of D-loop region sequence was carried out to estimate the genetic diversity and phylogenetic relationship within and among these three populations. Among 84 griffon specimens (44 Sardinian, 33 Cretan, and 7 Cypriot), we detected four haplotypes including a novel haplotype (HPT-D) that was exclusively found in the Cretan population with a frequency of 6.1%. When considered as a unique population, haplotype diversity (Hd) and nucleotide diversity (π) were high at 0.474 and 0.00176, respectively. A similar level of Hd and π was found in Sardinian and Cretan populations, both showing three haplotypes. The different haplotype frequencies and exclusivity detected were in accordance with the limited matrilineal gene flow (FST = 0.07097), probably related to the species reluctance to fly over sea masses. The genetic variability we observe today would therefore be the result of an evolutionary process strongly influenced by isolation leading to the appearance of island variants which deserve to be protected. Furthermore, since nesting sites and food availability are essential elements for colony settlement, we may infer that the island's colonization began when the first domestic animals were transferred by humans during the Neolithic. In conclusion, our research presents a first contribution to the genetic characterization of the griffon vulture populations in the Mediterranean islands of Sardinia, Crete and Cyprus and lays the foundation for conservation and restocking programs.