Florence Guignot

Effect of adiponectin on bovine granulosa cell steroidogenesis, oocyte maturation and embryo development.

BackgroundAdiponectin is an adipokine, mainly produced by adipose tissue. It regulates several reproductive processes. The protein expression of the adiponectin system (adiponectin, its receptors, AdipoR1 and AdipoR2 and the APPL1 adaptor) in bovine ovary and its role on ovarian cells and embryo, remain however to be determined.MethodsHere, we identified the adiponectin system in bovine ovarian cells and embryo using RT-PCR, immunoblotting and immunohistochemistry. Furthermore, we investigated in vitro the effects of recombinant human adiponectin (10 micro g/mL) on proliferation of granulosa cells (GC) measured by [3H] thymidine incorporation, progesterone and estradiol secretions measured by radioimmunoassay in the culture medium of GC, nuclear oocyte maturation and early embryo development.ResultsWe show that the mRNAs and proteins for the adiponectin system are present in bovine ovary (small and large follicles and corpus luteum) and embryo. Adiponectin, AdipoR1 and AdipoR2 were more precisely localized in oocyte, GC and theca cells. Adiponectin increased IGF-1 10(-8) M-induced GC proliferation (P < 0.01) but not basal or insulin 10(-8) M-induced proliferation. Additionally, adiponectin decreased insulin 10(-8) M-induced, but not basal or IGF-1 10(-8) M-induced secretions of progesterone (P < 0.01) and estradiol (P < 0.05) by GC. This decrease in insulin-induced steroidogenesis was associated with a decrease in ERK1/2 MAPK phosphorylation in GC pre-treated with adiponectin. Finally, addition of adiponectin during in vitro maturation affected neither the percentage of oocyte in metaphase-II nor 48-h cleavage and blastocyst day 8 rates.ConclusionsIn bovine species, adiponectin decreased insulin-induced steroidogenesis and increased IGF-1-induced proliferation of cultured GC through a potential involvement of ERK1/2 MAPK pathway, whereas it did not modify oocyte maturation and embryo development in vitro.

Relationships between post-mortem pH changes and some traits of sensory quality in veal

The aim of this study was to evaluate the relationships between the rate and extent of post-mortem pH changes and the colour, the cooking loss and the eating quality of veal. The experiment used 12 calves aged 18 weeks. Variations in ultimate pH were induced by adrenalin administration (0.1-0.4 mg/kg liveweight) to six of the animals. Measurements were made on the Longissimus thoracis muscle. pH and osmotic pressure were measured at 0.5 h, 4 h and 29 h after slaughter. Pigment content, drip loss and cooking loss were measured at 29 h after slaughter, and colour was measured at 2 days and 9 days after slaughter. Cooking loss, tenderness, juiciness and flavour of roasts were assessed at 9 days after slaughter. Correlations between colour traits and pH values were higher with ultimate pH than with pH at 0.5 h or 4 h after slaughter. Lightness, redness and reflectance decreased when the ultimate pH increased. Drip loss was correlated with the rate of pH fall (r = -0.80, P < 0.01 with pH at 4 h), while cooking loss was correlated with ultimate pH (r = -0.94, P < 0.01). Ultimate pH and the sensory quality traits were linearity and positively correlated (r = 0.83) for tenderness, 0.81 for juiciness and 0.71 for flavour, respectively).

Effect of PUFA on embryo cryoresistance, gene expression and AMPKα phosphorylation in IVF-derived bovine embryos

The objectives of the present study were to evaluate the effect of conjugated linoleic acid (CLA t10, c12, C18:2), linolenic acid (C18:3) and docosahexaenoic acid (DHA, C22:6) supplementation on in vitro bovine embryo development, embryo survival after cryopreservation, gene expression and AMPKalpha phosphorylation. Control groups with modified synthetic oviduct fluid (mSOF)+/-100microM beta-mercaptoethanol (beta-ME) were performed. The effects of co-culture with bovine oviduct epithelial cell (Boec) monolayers, serum supplementation and embryo development in the ewe oviduct, on gene expression were also examined. Experiments 1 and 2: a lower d 7 embryo survival was found with 100microM C22:6 and 100microM C18:2 supplementation compared to 1microM C22:6 and 100microM beta-ME supplementation (P<0.05). C18:3 supplementation had no effect on d 7 embryo survival, but 100microM C18:3 increased d 8 embryo survival compared to 100microM beta-ME supplementation (P<0.05). Experiments 3 and 4: stearoyl-CoA desaturase 1 (SCD1) and sterol regulatory element-binding transcription factor 1 (SREBP1) mRNA decreased after 10microM C22:6 supplementation compared to all other supplementations (P<0.05). A lower fatty acid desaturase 2 (FADS2) transcript level was found with 100microM C18:2, 10microM C22:6 and 10microM C18:3 supplementations compared to groups without fatty acid supplementation (P<0.05). Acetyl-CoA-carboxylase (ACC), fatty acid synthase (FAS), adipose differentiation-related protein (ADRP), acyl-CoA synthetase long-chain family member 1 (ACSL1), diacylglycerol O-acyltransferase 1 (DGAT1), carnitin palmitoyltransferase-II (CPT-II) mRNAs expression and AMPKalpha phosphorylation were not modified with PUFA supplementation. Experiment 5: SCD1 and FAS mRNA decrease in Boec group compared to serum supplementation, as SCD1 mRNA in ewe oviduct group (P<0.05). In conclusion, this study showed that a PUFA supplementation with C18:2, C18:3 or C22:6 in bovine culture development for 6 days and co-culture with Boec down-regulate mRNA expression of proteins involved in lipid metabolism in d 7-8 embryo (SCD1 and FADS2 desaturases), probably through SREBP1 mRNA regulation after 10microM C22:6 supplementation, indicating a modification of saturated/unsaturated fatty acid balance in bovine blastocyst.

Post mortem evolution of myofilament spacing and extracellular space in veal muscle.

The post mortem evolution of water distribution was studied in muscle tissue from veal calves in two experiments. Myofilament spacing, extracellular space and rate and extent of pH fall were determined in Psoas major muscle in Experiment 1. Extracellular space and rate and extent of pH fall were determined in Longissimus dorsi, Psoas major and Trapezius muscles in Experiment 2. In Experiment 2, the variability of ultimate pH was increased by using adrenalin injections. The myofilament spacing decreased after slaughter when pH reached values around 5·9. In both experiments, the extracellular space began to increase soon after slaughter in close relation with the pH changes. The size of the ultimate extracellular space was significantly correlated with the rate of pH fall, but not with the ultimate pH.

Bovine muscle 20S proteasome. II: Contribution of the 20S proteasome to meat tenderization as revealed by an ultrastructural approach

The role of the 20S proteasome proteolytic effects was revisited using an ultrastructural approach with the aim to explain some particular structural changes identified in type I muscles and in high pH meat. In both types of meat, major changes observed after ageing are an increase in the thickness of the Z-line followed by the appearance of an amorphous protein structure spreading out over the I-band. This was followed by a total degradation of this amorphous structure and of the Z-line. Partial transversal fragmentation of the myofibrils within the I-band can also be detected. The data reported clearly demonstrate that the 20S proteasome was able to mimic these sequential structural changes, a feature never obtained with either calpains or cathepsins. It is the first time that a direct implication of this complex in postmortem muscle is postulated.

Effect of different culture systems on adipocyte differentiation-related protein (ADRP) in bovine embryos

Bovine embryos cultured in serum-containing media abnormally accumulate lipid droplets, compared to their in vivo counterparts. The objective of this study was to investigate the effect of different culture systems on the mRNA expression and on the quantification and localisation of adipocyte differentiation-related protein (ADRP), a protein associated with lipid accumulation in bovine blastocysts. Two experiments were independently performed for ADRP mRNA expression analysis. In experiment A, blastocysts were produced in modified synthetic oviduct fluid (mSOF)+10% foetal calf serum (FCS), in coculture (bovine oviduct epithelial cells, Boec) and in ewe oviducts, whereas in experiment B, they were produced in mSOF+10μM docosahexaenoic acid (DHA) and in vivo. Control groups were also performed. ADRP mRNA expression was downregulated in the Boec, ewe oviduct and in vivo groups compared to the 10% FCS or DHA groups, respectively. Moreover, the expression of this protein was downregulated in the Boec group compared to the control group (P<0.05). A third experiment (experiment C) was performed to quantify and localise ADRP protein. Boec, in vivo and control groups were tested. After immunofluorescence staining followed by confocal microscopy analysis, embryonic ADRP was clearly localised around lipid droplets, indicating that ADRP is also a lipid droplet coat protein in bovine embryos. In conclusion, our results demonstrate that bovine embryos at the blastocyst stage expressed ADRP mRNA and protein, and that the embryonic culture system modified this expression.

Preimplantation genetic diagnosis in Welsh pony embryos after biopsy and cryopreservation

Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryopreserved embryos either 24 h after in vitro culture or after transfer to recipients. Fresh and nonbiopsied embryos were used as controls. Sex, coat color genes, myotony (neuromuscular disorder) diagnosis, and markers of parentage were investigated using PCR on biopsied cells after whole-genome amplification and on remaining embryos. The embryo survival rate after transfer was not affected by the micropipette biopsy (50%, = 8; 43%, = 7; and 50%, = 12, at d 30 for fresh biopsied embryos, vitrified biopsied embryos, and control embryos, respectively) but was significantly reduced by the use of microblade biopsy: 9 ( = 11) vs. 67% ( = 12) for control embryos. Successful sex determination was achieved for 82% ( = 28) of the micropipette biopsies and 100% ( = 50) of the microblade biopsies. Sex determined on biopsied cells was found to correspond completely (100%) with that determined on the remaining embryo ( = 37). More than 90% of the parentage checking markers, coat color, and myotony diagnosis were successfully determined on biopsies obtained with either a micropipette or a microblade. Mendelian incompatibility (7.5 and 5.5%) and embryo genotyping errors (6.6 and 8.6%) were low and not significantly different between the 2 methods. In conclusion, for the first time, pregnancy at Day 30 was obtained after transfer of Welsh pony biopsied and vitrified embryos >300 μm in diameter to recipient pony mares. The biopsied cells collected enabled multigenetic embryo diagnoses to be performed to a high degree of accuracy. The micropipette biopsy is the better method to apply on Welsh pony embryos.

Sex and PRNP genotype determination in preimplantation caprine embryos

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo.