Avian sperm-borne RNAs: optimisation of a new isolation protocol.

1. Sperm-borne RNAs are involved in sperm and embryonic protein translation, the regulation of early development and the epigenetic inheritance of the paternal phenotype. Sperm-borne RNA purification protocols generally include a cell purification stage to discard contamination by somatic cells. In avian species, no protocol is currently available to isolate all the populations composing sperm-borne RNAs.2. This study evaluated the presence of somatic cells in semen samples of chickens and quails using visual examination after fluorescent nuclei staining. The efficiency of somatic cell lysis buffer (SCLB) on chicken liver cells and its impacts on chicken sperm cell integrity was explored. Three different approaches were tested to isolate RNA: two developed for mammalian sperm cells and a commercial kit for somatic cells. The efficiency and reliability of each approach was determined based on RNA quality and purity. Eventually, the presence of miRNA and mRNA in purified avian sperm-borne RNAs was investigated by RT-(q)PCR.3. No somatic cells were found in chicken and quail semen. The SCLB totally lysed chicken liver cells but also induced sperm cell necrosis. Consequently, this treatment wasn't performed on samples prior to RNA isolation. Among the tested RNA purification protocols, the commercial one was the least variable and isolated RNA with the highest purity levels. No DNA contamination was observed. Furthermore, the samples contained miRNA and mRNA already known as present in mammalian sperm cells (gga-miR-100-5p, gga-miR-191-5p, GAPDH and PLCZ1), but mRNAs associated with leucocytes (CD4) and Sertoli cells (SOX4, CLDN11) were not detected. This protocol was successfully applied to quail sperm cells.4. Altogether, the study reveals that it is unnecessary to pre-treat samples to remove somatic cell contamination before RNA purification and successfully describes an isolation protocol for sperm-borne RNAs, including small non-coding and long coding RNAs, in two distinct avian species highly valuable as biological models.

Steroidome and metabolome analysis in gilt saliva to identify potential biomarkers of boar effect receptivity.

Optimal management of gilt reproduction requires oestrus synchronization. Hormonal treatments are used for this purpose, but there is a growing demand for non-hormonal alternatives, especially in organic farms. The boar effect is an important alternative opportunity to induce and synchronize oestrus without hormones. Before puberty, gilts exhibit a 'waiting period' during which boar exposure could induce and synchronize the first ovulation. We searched for salivary biomarkers of this period of boar effect receptivity to improve detection of the gilts to stimulate with the perspective of enhancing the efficacy of the boar effect. Saliva samples were collected from 30 Large-White×Landrace crossbred gilts between 140 and 175 days of age. Gilts were exposed twice a day to a boar and subjected to oestrus detection from 150 to 175 days of age. Among the 30 gilts, 10 were detected in oestrus 4 to 7 days after the first introduction of the boar and were considered receptive to the boar effect, 14 were detected in oestrus more than 8 days after first boar contact, and six did not show oestrus and were considered non-receptive. Saliva samples from six receptive and six non-receptive gilts were analyzed for steroidome and for metabolome using gas chromatography coupled to tandem mass spectrometry and 1H nuclear magnetic resonance spectroscopy, respectively. Four saliva samples per gilt were analyzed: 25 days and 11 days before boar introduction, the day of boar introduction, 3 days later for receptive gilts or 7 days later for non-receptive gilts. Twenty-nine steroids and 31 metabolites were detected in gilt saliva. Salivary concentrations of six steroids and three metabolites were significantly different between receptive and non-receptive gilts: progesterone and glycolate 25 days before boar introduction, 3α5ß20α- and 3ß5α20ß-hexahydroprogesterone, dehydroepiandrosterone, androstenediol, succinate, and butyrate 11 days before boar introduction, and 3ß5α-tetrahydroprogesterone on the day of boar introduction. Thus, nine potential salivary biomarkers of boar effect receptivity were identified in our experimental conditions. Further studies with higher numbers of gilts and salivary sampling points are necessary to ascertain their reliability.

Salivary and urinary metabolome analysis for pre-puberty-related biomarkers identification in porcine.

Estrus synchronization is important for optimal management of gilt reproduction in pig farms. Hormonal treatments, such as synthetic progestogens, are used on a routine basis, but there is a growing demand for non-hormonal alternative breeding tools. Before puberty, gilts exhibit a 'waiting period,' related to the ovarian development and gonadotrophin secretions, during which external stimulations, such as boar exposure, could induce and synchronize first ovulation. Practical non-invasive tools for identification of this period in farms are lacking. During this period, urinary oestrone levels are high, but urine sampling is difficult in group-housed females. The aim of this work was to search for specific biomarkers of the 'waiting period' in saliva and urine. In total, nine 144- to 147-day-old Large White gilts were subjected to trans-abdominal ultrasonography three times a week for 5 weeks until puberty detection (week -5 to week -1 before puberty). Urine and saliva samples were collected for oestrone assay to detect the 'waiting period' and for metabolome analysis using 1H-nuclear magnetic resonance spectroscopy to detect potential biomarkers of the 'waiting period.' Gilts were slaughtered 7 days after puberty detection for puberty confirmation. Results were consistent with ultrasonography data for six gilts. Urine and saliva samples from these six gilts were analyzed. Urinary estrone concentration significantly increased 2 weeks before puberty detection. Metabolome analysis of urine samples allowed the identification of 78 spectral bins, among them, 42 low-molecular-weight metabolites were identified. Metabolome analysis of salivary samples allowed the identification of 59 spectral bins, among them, 23 low-molecular-weight metabolites were detected and 17 were identified. No potential biomarker was identified in urinary samples. In saliva, butyrate and 2HOvalerate, 5.79 ppm (putatively uridine), formate, malonate and propionate could be biomarker candidates to ascertain the pre-puberty period in gilt reproduction. These results confirm that non-invasive salivary samples could allow the identification of the physiological status of the gilts and presumably the optimal time for application of the boar effect. This could contribute to synchronize puberty onset and hence to develop non-hormonal breeding tools.

A hybrid characterization framework to determine the visco-hyperelastic properties of a porcine zona pellucida.

The zona pellucida (ZP) is a specialized extracellular matrix surrounding the developing oocyte. This thick matrix consists of various types of glycoprotein that play different roles in the fertilization process. Nowadays, several techniques are available for assessing ZP's mechanical response. The basic assumption behind these methods is that the ZP behaves like an elastic body: hence, dissipative forces are neglected and Young's modulus remains unaffected by probe dynamics. However, dissipative forces are strongly regulated by the slippage of ZP chains past one another while reaction forces related to elastic deformations (driven by the ability of each chain to stretch) depend on the ZP structure (i.e. number of cross-links and distances between knots). Although viscous reaction forces generated by the ZP are one of the main factors regulating sperm transit, their peculiar behaviour along the ZP structure remains poorly understood and rarely investigated. In order to overcome this limitation, a novel visco-hyperelastic model describing the porcine ZP reaction forces generated by nanoindentations at different probe rates is developed and verified in this study. Visco-hyperelastic parameters of porcine ZP membranes are determined by means of a hybrid characterization framework combining atomic force microscopy nanoindentation measurements, nonlinear finite-element analysis and nonlinear optimization. Remarkably, it is possible to separate the contributions of hyperelastic and viscous terms to ZP mechanical response and evaluate the error made in the determination of ZP mechanical properties if viscous effects were not considered.

[Prescription of cholinesterase inhibitors in Alzheimer's disease in France in 2000-2001: an assessment of compliance with national guidelines for diagnosis and follow-up]. / Prescription des anticholinestérasiques dans la maladie d'Alzheimer en France en 2000-2001: évaluation du respect des procédures diagnostiques et de suivi.

INTRODUCTION: Alzheimer's disease (AD) became a treatable disease about ten years ago when cholinesterase inhibitors (CI) became available. We conducted a national population-based study in France in order to describe patients taking CI in 2001, to compare the diagnostic procedures of AD and the drug prescriptions with the French National guidelines, and to analyze the follow-up procedures of the patients. METHODS: The study was carried out in nine regions of France between December 1st 2000 and February 28th 2001 and included 3510 patients with CI treatment prescribed by the physicians affiliated with the National Social Security Health Care Fund. RESULTS: We found that the diagnostic procedures and the prescriptions were in compliance with the national guidelines with more than 90 percent of patients referred to a specialist, brain imaging in 85 percent and a MMSE scored between 10 and 26 in more than 90 percent. However, patient follow-up was in compliance with the guidelines in only 50 percent of patients. CONCLUSION: Diagnostic procedures of AD and first prescription of CI seem to be adequate in France, but patient follow-up needs to be improved.

[Value of the use of the oral route versus the injectable route for fluoroquinolones. Pharmacoeconomic incidence and bibliographical study]. / Intérêt de l'utilisation de la voie orale par rapport à la voie injectable pour les fluoroquinolones. Incidence pharmacoéconomique et étude bibliographique.

The aim of this study was to research the optimal conditions to shift to oral from injectable administration route for the fluoroquinolone antibiotics and the pharmacoeconomic and therapeutic impact of such a shift. Two indicators were used: proportion of the two administration routes, and mean cost per administration. The published results of pharmacokinetic studies in healthy and diseased subjects, and the clinical and/or pharmacokinetic studies including the notion of a therapeutic shift from the parenteral route to the oral route have been selected. The bioequivalence pharmacokinetic parameters of oral and injectable forms and the major clinical data of the therapeutic shift have been listed. Literature analysis reveals that there are few studies covering the specific assessment of the switch. The financial consequences of oral administration early use show the importance of such studies.

Actual usage of medical facilities by asthmatics in two French rural settings: a preliminary study.

Until recently the medical management of asthmatic patients with respect to their socio-cultural environment had only been superficially studied. Four hundred and fifty eight adult asthmatics were identified through a two-stage questionnaire in two rural districts of south west France. The study has mainly demonstrated: 1) the large number of subjects experiencing more than one attack per week (33% in Gironde, 40% in Lot et Garonne); 2) the large number of subjects feeling disabled in their occupational and day-to-day life (52% in Gironde, 54% in Lot et Garonne); 3) the absence of a relationship between the severity of the disease and the use of medical facilities in the community. Despite the frequency of the attacks and the perceived disability, 27% of the patients felt that their asthma did not require any treatment and up to 60% did not seek medical help even during an attack; 4) the role of population density, type of dwelling and social status in the management of asthma. We conclude that under-treatment is widespread in these two rural settings and is closely related with the association of at least two of these three characteristics: 1) living in a low population density area; 2) living in an isolated dwelling; and 3) being a farm owner.