Stefan Deleuze

Cysteamine supplementation of in vitro maturation media: a review.

Under in vitro culture conditions, oxidative modifications of cell components via increased reactive oxygen species (ROS) represent a major culture induced stress. Anti-oxidant systems such as glutathione (GSH) can attenuate the deleterious effects of oxidative stress by scavenging ROS. It has been suggested that GSH content in oocytes may serve as a reservoir protecting the zygote and the early embryos from oxidative damage before genomic activation and de novo GSH synthesis occur. Addition of low molecular weight compounds to culture media, such as cysteamine, can increase GSH levels by increasing cysteine uptake. Quite naturally, effects of supplementation of in vitro maturation (IVM) media with low molecular weight thiols have been studied in various species. This article reviews the use of cysteamine supplementation for IVM, its effects on maturation rates and further embryo development.

Changes in histone H4 acetylation during in vivo versus in vitro maturation of equine oocytes

Epigenetic modifications are established during gametogenesis and preimplantation embryonic development. Any disturbance of the normal natural environment during these critical phases could cause alterations of the epigenetic signature. Histone acetylation is an important epigenetic modification involved in the regulation of chromatin organization and gene expression. The present study was aimed to determine whether the proper establishment of post-translational histone H4 acetylation at lysine 8 (AcH4K8), 12 (AcH4K12) and 16 (AcH4K16) of equine oocytes is adversely affected during in vitro maturation (IVM) when compared with in vivo matured oocytes collected from naturally cycling mares not undergoing ovarian hyperstimulation. The acetylation patterns were investigated by means of indirect immunofluorescence staining with specific antibodies directed against the acetylated lysine residues. Our results indicate that the acetylation state of H4 is dependent on the chromatin configuration in immature germinal vesicle (GV) stage oocytes and it changes in a residue-specific manner along with the increase of chromatin condensation. In particular, the levels of AcH4K8 and AcH4K12 increased significantly, while AcH4K16 decreased significantly from the fibrillar to the condensed state of chromatin configuration within the GV. Moreover, during meiosis, K8 and K12 were substantially deacetylated without any differences between in vivo and in vitro conditions, while K16 displayed a strong acetylation in oocytes matured in vivo, and in contrast, it was markedly deacetylated following IVM. Although the functional meaning of residue-specific acetylation during oocyte differentiation and meiotic resumption needs further investigation, our results support the hypothesis that IVM conditions can adversely affect oocyte ability to regulate the epigenetic reprogramming, critical for successful meiosis and subsequent embryonic development.

The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved?

BackgroundOviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse.Methods & resultsIn the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1) gametes co-incubated with equine vs porcine OEC, 2) intact cumulus-oocyte complexes vs denuded oocytes, 3) OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4) in vivo vs in vitro matured oocytes.In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A) in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though osteopontin slightly increased the IVF rates.ConclusionOur study shows a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates, though the rates remain low. Furthers studies are necessary to identify the proteins involved. We showed that the surface plasmon resonance technique is efficient and powerful to analyze molecular interactions during fertilization.

Efficiency of embryonic development after intrafollicular and intraoviductal transfer of in vitro and in vivo matured horse oocytes.

In vivo techniques, such as intraoviductal oocyte transfer (OT) and intrafollicular oocyte transfer (IFOT), can be considered as alternatives to bypass the lack of efficient superovulation treatments and the inadequacy of conventional in vitro fertilization techniques in the horse. We compared embryo production after transfer of in vivo recovered oocytes (1) into a recipients oviduct or (2) into her preovulatory follicle either immediately after ovum pick-up or (3) after in vitro maturation (IVM). Recipients were inseminated with fresh semen of a stallion with a known normal fertility. Ten days after surgery, rates of embryos collected in excess to the number of ovulations were calculated and compared for each group. Embryo collection rates were 32.5% (13 of 40), 5.5% (3 of 55), and 12.8% (6 of 47) for OT, post-IVM IFOT, and immediate IFOT, respectively. Oocyte transfer significantly yielded more embryos than did immediate IFOT and post-IVM IFOT. We also showed that in vitro matured oocytes could successfully be used for IFOT. Our results also suggest that improvement of the IFOT technique could turn it into an inexpensive and easy-to-perform procedure that could be an answer to the inefficiency of superovulation treatments in the mare.

Influence of cysteamine on in vitro maturation, in vitro and in vivo fertilization of equine oocytes.

Contents The effect of cysteamine on in vitro nuclear and cytoplasmic maturation of equine oocytes collected by transvaginal ultrasound guided follicular aspiration was assessed. Oocytes were matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining, penetration rates after two different in vitro fertilization (IVF) techniques (IVF media with ionophore and Hepes buffer with heparin) and the embryo yield following oocyte intra-oviductal transfer were used as a criterion for assessing nuclear and cytoplasmic maturation, respectively. Contrary to the data described in other domestic species, there was no effect of cysteamine on in vitro nuclear maturation, IVF or in vivo embryonic development under our conditions. Ovum pick up yields (52%) and maturation rates (control group: 47% and cysteamine group: 55%) were similar to those previously reported. From 57 oocytes transferred to the oviduct in each group, the number of embryos collected was 10 (17%) in the control group and five in the cysteamine group (9%). Those two percentages were not statistically different (p > 0.05). No effect of IVF technique was seen on the success rate (6%) in each group.

Effect of breed, sex, age and body weight on Echocardiographic Measurements in the Equine Species

Little is known about the effect of various animals signalment variables on echocardiographic reference values in the equine species. This study was performed to evaluate the effect of sex, breed, age and body weight (BW) on echocardiographic measurements in the equine species. Echocardiography was performed on 212 ponies or horses of various breeds, aged from 1 day to 37 years old (mean±SD: 7.8 ± 5.8 years), BW 38-890 kg (mean ± SD: 421 ± 133 kg), and free of cardiac disease. Fifty of those animals aged from 2 months to 35 years old (mean ± SD: 11.6 ± 6.4 years old); BW 77-662 kg (mean ± SD: 436 ± 135 kg) were also examined using the pulsed-wave Doppler mode. Standard two-dimensional and M-mode echocardiography were performed on all animals. Standard pulsed-wave Doppler examination of each cardiac valve was performed on the 50 first examined animals. Data were analysed using a general linear model including the effect of sex, age, breed and BW after logarithmic transformation of the data. Therefore, the same analysis was performed separately on animals aged ≤ 2 years-old and on older animals. All dimensional echocardiographic measurements were significantly affected by BW and most of them were significantly affected by breed, but not by sex. Only the aortic and the pulmonary artery internal diameter were significantly affected by age. None of the Doppler measurements were significantly affected by the tested variables. In conclusion, in the equine species, dimensional echocardiographic reference values should be established using regression equations as a function of BW, which could increase the diagnostic value of this leading technique in equine cardiology. Breed could also have an effect on those measurements.

Two-dimensional and M-mode echocardiographic reference values in healthy adult Saanen goats

Echocardiography has become a routine non-invasive cardiac diagnostic tool in most species. Accurate measurement of cardiac dimensions requires reference values, which are poorly documented in goats. The aim of the present study was to test the inter-day repeatability and to establish the reference values of two-dimensional (2D-) and time-motion (M-) mode echocardiographic variables in healthy adult Saanen goats. Six goats were investigated three times by the same observer at one-day interval using a standardised 2D- and M-mode echocardiographic protocol. The intra-observer inter-day repeatability was tested using analysis of variance, calculation of the coefficient of variation and confidence intervals. A single echocardiographic examination was performed in six other goats, and values obtained in the 12 goats were used to establish the 2D- and M-mode echocardiographic reference values in healthy adult female Saanen goats. Statistical analysis revealed a good inter-day repeatability of the echocardiographic cardiac measurements. Echocardiographic reference values obtained in healthy adult Saanen goats seemed slightly higher than those reported in healthy Swedish domestic goats and were similar to those reported in healthy adult sheep.

Sperm quality analysis in XX, XY and YY males of the Nile tilapia (Oreochromis niloticus)

In Nile tilapia (Oreochromis niloticus), individuals with atypical sexual genotype are commonly used in farming (use of YY males to produce all-male offspring), but they also constitute major tools to study sex determinism mechanisms. In other species, sexual genotype and sex reversal procedures affect different aspects of biology, such as growth, behavior and reproductive success. The aim of this study was to assess the influence of sexual genotype on sperm quality in Nile tilapia. Milt characteristics were compared in XX (sex-reversed), XY and YY males in terms of gonadosomatic index, sperm count, sperm motility and duration of sperm motility. Sperm motility was measured by computer-assisted sperm analysis (CASA) quantifying several parameters: total motility, progressive motility, curvilinear velocity, straight line velocity, average path velocity and linearity. None of the sperm traits measured significantly differed between the three genotypes. Mean values of gonadosomatic index, sperm concentration and sperm motility duration of XX, XY and YY males, respectively ranged from 0.92 to 1.33%, from 1.69 to 2.22 ×10(9) cells mL(-1) and from 1804″ to 2732″. Mean values of total motility and curvilinear velocity 1 min after sperm activation, respectively ranged from 53 to 58% and from 71 to 76 μm s(-1) for the three genotypes. After 3 min of activity, all the sperm motility and velocity parameters dropped by half and continued to slowly decrease thereafter. Seven min after activation, only 9 to 13% of spermatozoa were still progressive. Our results prove that neither sexual genotype nor hormonal sex reversal treatments affect sperm quality in male Nile tilapias with atypical sexual genotype.

Repeatability, variability and reference values of pulsed wave Doppler echocardiographic measurements in healthy Saanen goats

BackgroundPulsed wave (PW) Doppler echocardiography has become a routine non invasive cardiac diagnostic tool in most species. However, evaluation of intracardiac blood flow requires reference values, which are poorly documented in goats. The aim of this study was to test the repeatability, the variability, and to establish the reference values of PW measurements in healthy adult Saanen goats. Using a standardised PW Doppler echocardiographic protocol, 10 healthy adult unsedated female Saanen goats were investigated three times at one day intervals by the same observer. Mitral, tricuspid, aortic and pulmonary flows were measured from a right parasternal view, and mitral and aortic flows were also measured from a left parasternal view. The difference between left and right side measurements and the intra-observer inter-day repeatability were tested and then the reference values of PW Doppler echocardiographic parameters in healthy adult female Saanen goats were established.ResultsAs documented in other species, all caprine PW Doppler parameters demonstrated a poor inter-day repeatability and a moderate variability. Tricuspid and pulmonary flows were best evaluated on the right side whereas mitral and aortic flows were best obtained on the left side, and reference values are reported for healthy adult Saanen goats.ConclusionsPW Doppler echocardiography allows the measurement of intracardiac blood flow indices in goats. The reference values establishment will help interpreting these indices of cardiac function in clinical cardiac cases and developing animal models for human cardiology research.

Descriptive Study of 32 Cases of Doxycycline-Overdosed Calves

BACKGROUNDnReports of doxycycline-induced toxicity are limited despite common use of this antibiotic to treat infectious respiratory disorders in calves.nnnOBJECTIVEnTo describe previously unreported kidney lesions and diagnostic test results in doxycycline-overdosed calves and to compare these results with other findings reported previously.nnnANIMALSnThirty-two calves that presented with adverse effects after receiving high doses of doxycycline as a treatment for mild respiratory disorders.nnnMETHODnRetrospective review of medical records.nnnRESULTSnClinical examination identified mainly lethargy, dyspnea, cough, tongue paresia or paralysis associated with dysphagia and sialorrhea, tachycardia, tachypnea, and signs of myopathy. Blood analysis indicated increases in creatine kinase, lactate dehydrogenase, aspartate aminotransferase, and sorbitol dehydrogenase activities and increased serum creatinine and urea concentrations. ECG recordings and Doppler echocardiography examination identified ventricular premature beats and a decrease in left ventricular global and systolic function, respectively. Necropsy and histopathology disclosed necrosis of the myocardium, tongue, and some striated muscles, acute renal tubular necrosis, and fatty degeneration or congestion of the liver.nnnCONCLUSIONSnMost of these findings corroborate previous observations made in doxycycline-overdosed calves, and further suggest myocardial and striated muscular toxicity as well as renal toxicity in doxycycline-overdosed calves.