Neonatal mortality in dogs : prognostic value of Doppler ductus venosus waveform evaluation - Preliminary results

Aim: To define the prognostic value of Doppler ultrasonographic morphology of ductus venosus (DV) waveform on canine neonatal mortality. Materials and Methods: Fifty-four healthy pregnant bitches underwent fetal ultrasonographic assessment. The DV waveforms were classified as diphasic (dDVw) or triphasic (tDVw) and compared with neonatal mortality. Results: Ninety-three fetuses were evaluated. Twenty fetuses belonged to litters with neonatal mortality, in which tDVw was observed. Seven fetuses belonged to litters without neonatal mortality, in which tDVw was observed. Fifty-eight fetuses belonged to litters without neonatal mortality, in which only dDVw was observed. Eight fetuses belonged to litters with neonatal mortality, in which only dDVw was observed. The correlation between tDVw and neonatal mortality was statistically significant (odds ratio [OR], 20.7; p<0.0001). Considering only pregnancies with one or two fetuses with the same DV waveform: Two fetuses with tDVw belonged to litters with neonatal mortality; 1 foetus with tDVw belonged to litter without neonatal mortality and 26 fetuses showed dDVw without neonatal mortality. The correlation between tDVw and neonatal mortality even in litters up to two pups was statistically significant (OR, 88.3; p=0.01). Conclusion: Echo-Doppler assessment of DV is feasible in canine fetuses, and the presence tDVw seems to be related to neonatal mortality

Asymptomatic unilateral ovarian leiomioma in a German shepherd bitch

This report shows for the first time clinical imaging (ultrasound and computed tomography), histological and immunohistochemical findings of an ovarian leiomyoma, coincidentally diagnosed in an asymptomatic unmated nulliparous ten year-old German shepherd bitch concurrently suffering from multiple mammary tumors. A thorough examination allowed the differentiation of ovarian leiomyoma from other spindle cell tumors. An accurate description of the diagnostic procedures useful in the managing of ovarian leiomyoma could provide valuable information to veterinary practitioners. Indeed, despite its rarity and nonspecific symptoms, ovarian leiomyoma may also affect the dog with an unknown potential risk

The methylome of the hypothalamus of prepubertal and pubertal goats

Puberty is the fulfillment of fertility, a process involving physiological and morphological development. It is well known that the increased hypothalamic secretion of the gonadotropin-releasing hormone (GnRH) is essential for the activation of this process, even if the elements coordinating the timing of puberty have not been fully identified1,2. Recent studies provide proof that there is an epigenetic regulation of female puberty, and DNA methylation, the most studied epigenetic modification, plays a major role in it3. We analyzed DNA methylation patterns of 5 Alpine goats at their prepubertal stage and 5 that reached puberty in order to highlight differences in their methylome. Detection of methylated regions across the goat genome involved a Methyl Binding Domain (MBD) enrichment followed by deep sequencing (Hiseq2000 Illumina). The software ChIPseeqer4 permitted the identification of peaks corresponding to hyper-methylated regions. We have observed a higher methylation level in prepubertal goats. The distribution of the methylation peaks across the genome and within CpG islands per chromosome per group of animals has been analyzed. Furthermore, we have investigated differential methylation in genes associated with puberty. Specifically, Cbx7, coding for a core component of the Polycomb group silencing complex, and GnRHR, the gene coding for GnRH receptor, showed a higher number of peaks into two intragenic fragments within prepubertal goats. These results, accompanied by transcriptome analysis, provide a foundation for elucidating the role of DNA methylation in the complex mechanisms that drive puberty in goat species

A first glance on the epigenome of Capra hircus

DNA methylation and microRNAs (miRNA) are two important forms of epigenetic modifications that play an important role in gene regulation in animals. Methylation at the carbon 5 position of cytosine residues is a fundamental layer of cellular differentiation through the control of transcriptional potential. MiRNA are small noncoding RNA molecules that regulate gene expression. Complete DNA methylomes for several organisms are now available; at the present, methylome of the domestic goat is unexplored. There is also still limited knowledge about miRNAs expression profiles in small ruminant species. Therefore, to contribute information on epigenetic modification in Capra hircus, we analysed the methylome and the miRNA population of three tissues (hypothalamus, pituitary and ovary) from 3 adult Saanen goats. We used Methylated DNA binding domain sequencing with enrichment of methylated DNA fragments and next generation sequencing. We produced least 23 million reads per sample, which were aligned to the goat reference genome. Further analyses were performed to identify peaks corresponding to hyper-methylated regions. We sequenced miRNAs expressed in the three tissues with Illumina high-throughput sequencing. Reads were mapped on the Capra hircus reference genome and both known and novel miRNAs, and miRNA target sites were identified using information collected in miRBase and using specific bioinformatic tools. This study produced a comprehensive miRNA profile related to the biology of goat. Furthermore, this is the first work dealing with methylome in Capra hircus: our preliminary results could provide new information for a deeper comprehension of epigenetic mechanisms of this species

Nefopam, an analogue of orphenadrine, protects against both NMDA receptor-dependent and independent veratridine-induced neurotoxicity

Nefopam hyghochloride is a potent analgesic compound commercialized in most Western Europe for 20 years, which possesses a profile distinct from that of opioids or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. While, nefopam structure resembles that of orphenadrine, an uncompetitive NMDA receptor antagonist, here we report that differently from orphenadrine, nefopam (100 microM) failed to protect cultured cerebellar neurons from excitotoxicity following direct exposure of neurons to glutamate. Moreover, nefopam failed to displace MK-801 binding to hippocampal membranes. Nefopam effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. The later phase (24 h) of neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. Nefopam effect was not mimicked by the GABA receptor agonist muscimo

Novel effect of nefopam preventing cGMP increase, oxygen radical formation and neuronal death induced by veratridine

Nefopam hydrochloride is a potent analgesic compound that possesses a profile distinct from that of opiods or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. Here we have used cultured cerebellar neurons to test the hypothesis that nefopam may modulate voltage sensitive sodium channel (VSSC) activity. Nefopam (100 microM) effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the VSSC activator veratridine. Delayed neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. In contrast, excitotoxicity following direct exposure of neurons to glutamate was not affected. Neuroprotection by nefopam was dose-dependent. 50% protection was obtained at 57 microM while full neuroprotection was achieved at 75 microM nefopam. Veratridine-induced sodium influx was completely abolished in nefopam-treated neurons. Intracellular cGMP and oxygen radical formation following VSSC stimulation by veratridine were also effectively prevented by nefopam. Our data are consistent with an inhibitory action of nefopam on VSSC and suggest that nefopam may modulate the release of endogenous glutamate following activation of these channels. This novel action of nefopam may be of great interest for the treatment of neurodegenerative disorders involving excessive glutamate release and neurotransmission

Sox9 Duplications Are a Relevant Cause of Sry-Negative XX Sex Reversal Dogs

Sexual development in mammals is based on a complicated and delicate network of genes and hormones that have to collaborate in a precise manner. The dark side of this pathway is represented by pathological conditions, wherein sexual development does not occur properly either in the XX and the XY background. Among them a conundrum is represented by the XX individuals with at least a partial testis differentiation even in absence of SRY. This particular condition is present in various mammals including the dog. Seven dogs characterized by XX karyotype, absence of SRY gene, and testicular tissue development were analysed by Array-CGH. In two cases the array-CGH analysis detected an interstitial heterozygous duplication of chromosome 9. The duplication contained the SOX9 coding region. In this work we provide for the first time a causative mutation for the XXSR condition in the dog. Moreover this report supports the idea that the dog represents a good animal model for the study of XXSR condition caused by abnormalities in the SOX9 locus

The DNA Methylation Pattern of Prepubertal and Pubertal Alpine Goats

Puberty timing is controlled by many genes and the elements coordinating this process have not completely been identified. Hypothalamus is a pivotal organ in the control of sexual maturation. There is evidence that epigenetic modifications, such as DNA methylation, play a key role in the process. The methylome of the hypothalamus of 10 Alpine goats, 5 at a prepubertal stage (93\ub18 days old) and 5 at their pubertal stage (230\ub117 days old) was analysed to investigate the differences at the DNA methylation level behind these physiological changes. In order to evaluate differentially methylated regions, Methylated DNA Binding Domain sequencing (MBD-seq) with enrichment of methylated DNA fragments and next generation sequencing (Hiseq2000 Illumina) were performed. An average of 24,13 million of reads (range 18.00 and 30.11 million of reads) were produced per sample and peaks corresponding to hyper-methylated regions were estimated using the software ChIPseeqer. The analysis showed that there was an increase in methylation before puberty.The extent of methylation had a median value (\ub1IQR) of 12.32\ub110.21 Mbp of the genome for prepubertal goats, compared with 8.18\ub19.71 Mbp for pubertal goats. Significantly increased methylation was seen on 11 chromosomes in prepubertal goats. Among these, chromosomes 4 and 7 were the most highly significant differentially methylated. In showing that female puberty in goats is associated with amodification of the DNA methylation pattern in the hypothalamus, these results add information on the complex mechanisms that control puberty in mammals